Differentiation of signals for target nucleic acid sequences

ABSTRACT

The present invention relates to differentiating signals of interest for target nucleic acid sequences. The present invention permits to obtain an individual signal value (i.e., variable) contained in a total signal detected at detection temperatures by using mathematical equations. The present invention based on equation-solving approach enables to obtain the individual signal value in a systematical manner, thereby providing analysis results in much more accurate and convenient manner.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of PCT/KR2015/013461, filed on Dec.9, 2015, which claims priority to U.S. Patent Application No.62/154,319, filed on Apr. 29, 2015, which claims priority to U.S. PatentApplication No. 62/089,723, filed on Dec. 9, 2014, the entire contentsof each of which are hereby incorporated in total by reference.

SEQUENCE LISTING

This application incorporates by reference the Sequence Listingcontained in an ASCII text file named “361406_0038_SeqList.txt”submitted via EFS-Web. The text file was created on Jun. 9, 2017, and is4 KB in size.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to differentiating signals of interest fortarget nucleic acid sequences.

Description of the Related Art

For detection of target nucleic acid sequences, real-time detectionmethods are widely used to detect target nucleic add sequences withmonitoring target amplification in a real-time manner. The real-timedetection methods generally use labeled probes or primers specificallyhybridized with target nucleic acid sequences. The exemplified methodsby use of hybridization between labeled probes and target nucleic addsequences include Molecular beacon method using dual-labeled probes withhairpin structure (Tyagi et al, Nature Biotechnology v.14 Mar. 1996),HyBeacon method (French D J et al., Mol. Cell Probes, 15(6):363-374(2001)), Hybridization probe method using two probes labeled each ofdonor and acceptor (Bernad et al, 147-148 Clin Chem 2000; 46) and Luxmethod using single-labeled oligonucleotides (U.S. Pat. No. 7,537,886).TaqMan method (U.S. Pat. Nos. 5,210,015 and 5,538,848) usingdual-labeled probes and its cleavage by 5′-nuclease activity of DNApolymerase is also widely employed in the art.

The exemplified methods using labeled primers include Sunrise primermethod (Nazarenko et al, 2516-2521 Nucleic Acids Research, 1997, v.25no. 12, and U.S. Pat. No. 6,117,635), Scorpion primer method (Whitcombeet al, 804-807, Nature Biotechnology v.17 Aug. 1999 and U.S. Pat. No.6,326,145) and TSG primer method (WO 2011/078441).

As alternative approaches, real-time detection methods using duplexesformed depending on the presence of target nucleic acid sequences havebeen proposed: Invader assay (U.S. Pat. Nos. 5,691,142, 6,358,691 and6,194,149), PTOCE (PTO cleavage AND extension) method (WO 2012/096523),PCE-SH (PTO Cleavage and Extension-Dependent Signaling OligonucleotideHybridization) method (WO 2013/115442), PCE-NH (PTO Cleavage andExtension-Dependent Non-Hybridization) method (PCT/KR2013/012312).

The conventional real-time detection technologies described above detectsignals generated from fluorescent labels at a selected detectiontemperature in signal amplification process associated with or with notarget amplification. When a plurality of target nucleic add sequencesusing a single type of label in a single reaction tube are detected inaccordance with the conventional real-time detection technologies,generated signals for target nucleic acid sequences are notdifferentiated from each other. Therefore, the conventional real-timedetection technologies generally employ different types of labels fordetecting a plurality of target nucleic acid sequences. The meltinganalysis using T_(m) difference permits to detect a plurality of targetnucleic acid sequences even a single type of label. However, the meltinganalysis has serious shortcomings in that its performance time is longerthan real-time technologies and design of probes with different T_(m)values becomes more difficult upon increasing target sequences.

Accordingly, where novel methods or approaches being not dependent onmelting analysis for detecting a plurality of target nucleic addsequences using a single type of label in a single reaction vessel and asingle type of detector are developed, they enable to detect a pluralityof target nucleic add sequences with dramatically enhanced convenience,cost-effectiveness and efficiency. In addition, the combination of thenovel methods with other detection methods (e.g., melting analysis)would result in detection of a plurality of target nucleic acidsequences using a single type of label in a single reaction vessel withdramatically enhanced efficiency.

Throughout this application, various patents and publications arereferenced and citations are provided in parentheses. The disclosure ofthese patents and publications in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

The present inventors have made intensive researches to develop novelmethods for qualitatively or quantitatively detecting a target nucleicacid sequence, particularly a plurality of target nucleic add sequencesin more accurate and convenient manner. As a result, we have found thatsignals for target nucleic acid sequences are obtained at detectiontemperatures and then detection results are processed by using suitableequations, thereby enabling to detect a plurality of target nucleic acidsequences even using a single type of label in a single reaction vesseland a single type of detector with dramatically enhanced convenience,cost-effectiveness and efficiency.

Accordingly, it is an object of this invention to provide a method and akit for differentiating signals of interest for each of two targetnucleic acid sequences comprising a first target nucleic acid sequence(T1) and a second target nucleic acid sequence (T2) in a sample, whichare not differentiable by a single type of detector.

It is another object of this invention to provide a method and a kit fordifferentiating signals of interest for each of target nucleic acidsequences in the number of N in a sample, which are not differentiableby a single type of detector.

It is still another object of this invention to provide a computerreadable storage medium containing instructions to configure a processorto perform a method for differentiating signals of interest for each oftwo target nucleic acid sequences comprising a first target nucleic addsequence (T1) and a second target nucleic acid sequence (T2) in asample, which are not differentiable by a single type of detector.

It is further object of this invention to provide a computer readablestorage medium containing instructions to configure a processor toperform a method for differentiating signals of interest for each oftarget nucleic acid sequences in the number of N in a sample, which arenot differentiable by a single type of detector.

It is still further object of this invention to provide a device fordifferentiating signals of interest for each of two target nucleic acidsequences comprising a first target nucleic acid sequence (T1) and asecond target nucleic acid sequence (T2) in a sample, which are notdifferentiable by a single type of detector.

It is another object of this invention to provide a device fordifferentiating signals of interest for each of target nucleic addsequences in the number of N in a sample, which are not differentiableby a single type of detector.

It is still another object of this invention to provide a computerprogram to be stored on a computer readable storage medium to configurea processor to perform a method for differentiating signals of interestfor each of two target nucleic add sequences comprising a first targetnucleic add sequence (T1) and a second target nucleic acid sequence (T2)in a sample, which are not differentiable by a single type of detector.

It is further object of this invention to provide a computer program tobe stored on a computer readable storage medium to configure a processorto perform a method for differentiating signals of interest for each oftarget nucleic acid sequences in the number of N in a sample, which arenot differentiable by a single type of detector.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A represents the detection results of a first target nucleic acidsequence (genome DNA of Neisseria gonorrhoeae, NG), a second targetnucleic acid sequence (genome DNA of Chlamydia trachomatis, CT) andtheir combination at a first detection temperature (60° C.) and a seconddetection temperature (72° C.). The signals for NG and CT were generatedby the TaqMan probe method. NTC denotes no template control.

FIG. 1B represents obtaining reference values based on the detectionresults of FIG. 1A. The ratio of the signal at the first detectiontemperature to the signal at the second detection temperature is one ofexemplified reference values.

FIG. 1C represents plotting results for S_(T1D1) and S_(T2D1) obtainedby solving the simultaneous equation 1. Experimentally obtainedreference values were used. The dotted line represents a threshold whichwas determined with referring to the result of NG only sample and CTonly sample in FIG. 1A to verify the significance of the obtainedamplification curves.

FIG. 1D represents plotting results for S_(T1D2) and S_(T2D2) obtainedby solving the simultaneous equation 2. Experimentally obtainedreference values were used. The dotted line represents a threshold.

FIG. 2A represents the detection results of a first target nucleic acidsequence (genome DNA of Neisseria gonorrhoeae, NG), a second targetnucleic acid sequence (genome DNA of Chlamydia trachomatis, CT) andtheir combination at a first detection temperature (60° C.) and a seconddetection temperature (72° C.). The signals for NG and CT were generatedby the PTOCE method. The signal generating means for CT was designed toprovide a signal of interest at 60° C. and 72° C. and those for NG wasdesigned to provide a signal of interest at only 60° C.

FIG. 2B represents obtaining reference values based on the detectionresults of FIG. 2A. The ratio of the signal at the first detectiontemperature to the signal at the second detection temperature is one ofexemplified reference values.

FIG. 2C represents plotting results for S_(T1D1) and S_(T2D1) obtainedby solving the equation set 2 (S_(T1D2)=0). The dotted line represents athreshold which was determined with referring to the result of NG onlysample and CT only sample in FIG. 2A to verify the significance of theobtained amplification curves.

FIG. 2D represents plotting results for S_(T1D2) and S_(T2D2) obtainedby solving the equation set 2 (S_(T1D2)=0). The dotted line represents athreshold.

FIG. 2E represents plotting results for S_(T1D1) and S_(T2D1) obtainedby solving the equation set 3 in which experimentally obtained referencevalues were used. The dotted line represents a threshold.

FIG. 2F represents plotting results for S_(T1D2) and S_(T2D2) obtainedby solving the equation set 3 in which experimentally obtained referencevalues were used. The dotted line represents a threshold.

FIG. 2G represents plotting results for S_(T1D1) and S_(T2D1) obtainedby solving the equation set 4 in which an arbitrary-selected constantvalue was used as RV_(T1D1/D2). The dotted line represents a threshold.

FIG. 2H represents plotting results for S_(T1D2) and S_(T2D2) obtainedby solving the equation set 4 in which an arbitrary-selected constantvalue was used as RV_(T1D1/D2) The dotted line represents a threshold.

FIGS. 3A and 3B represent the detection results of three target nucleicacid sequences [genomic DNA of Neisseria gonorrhoeae (NG), genomic DNAof Chlamydia trachomatis (CT), and genomic DNA of Mycoplasma genitalium(MG)] and their combination at three detection temperature (60° C., 72°C. and 95° C.). The signals for NG and CT were generated by the TOCEmethod, and the signals for MG were generated by the TaqMan probemethod.

FIG. 3C represents obtaining reference values based on the detectionresults of FIG. 3A. The ratio of the signal between detectiontemperatures is one of exemplified reference values.

FIGS. 3D and 3E represents plotting results for S_(T1D1), S_(T2D1) andS_(T3D1) obtained by solving the simultaneous equation 5. Experimentallyobtained reference values were used. The dotted line represents athreshold which was determined with referring to the result of NG onlysample, CT only sample and MG only sample in FIG. 3A to verify thesignificance of the obtained amplification curves.

FIGS. 3F and 3G represents plotting results for S_(T1D2), S_(T2D2) andS_(T3D2) obtained by solving the simultaneous equation 6. Experimentallyobtained reference values were used. The dotted line represents athreshold.

FIGS. 3H and 3I represents plotting results for S_(T1D3), S_(T2D3) andS_(T3D3) obtained by solving the simultaneous equation 7. Experimentallyobtained reference values were used. The dotted line represents athreshold.

DETAILED DESCRIPTION OF THIS INVENTION

The most prominent feature of the present invention is detect aplurality of target nucleic acid sequences even using a single type oflabel and a single type of detector in a signal reaction vessel. Thepresent invention employing different detection temperatures andmathematical equations for solving variables enables to detect aplurality of target nucleic acid sequences even with a single type oflabel in a single reaction vessel. The elements of the present inventionare selected in compliance with the feature of the present invention andfabricated into a surprising process for detect target nucleic acidsequences.

Conventional real-time PCR methods require two types of fluorescentlabels or melting analysis for detection of two target nucleic acidsequences in a single reaction vessel.

The present invention permits real-time PCR protocols to detect twotarget nucleic add sequences even using a single type of fluorescentlabel in a single reaction vessel.

I. Differentiation of Signals of Interest for Two Target Nucleic AcidSequences

In one aspect of this invention, there is provided a method fordifferentiating signals of interest for each of two target nucleic acidsequences comprising a first target nucleic add sequence (T1) and asecond target nucleic acid sequence (T2) in a sample, which are notdifferentiable by a single type of detector, comprising:

(a) incubating the sample with a first signal-generating means fordetection of the first target nucleic add sequence (T1) and a secondsignal-generating means for detection of the second target nucleic acidsequence (T2) and detecting signals at a first detection temperature(D1) and a second detection temperature (D2); wherein the signals ofinterest to be generated by the two signal-generating means are notdifferentiated for each target nucleic add sequence by a single type ofdetector;

(b) providing the following two equations each of which comprisesvariables representing the signals of interest generated at eachdetection temperature for the two target nucleic acid sequences;S _(T1D1) +S _(T2D1) =S _(D1)  (I)S _(T1D2) +S _(T2D2) =S _(D2)  (II)

wherein (S_(D1)) is a signal detected at the first detectiontemperature, (S_(D2)) is a signal detected at the second detectiontemperature; (S_(T1D1)) is a variable representing a signal of interestgenerated by the first signal-generating means at the first detectiontemperature, (S_(T2D1)) is a variable representing a signal of interestgenerated by the second signal-generating means at the first detectiontemperature, (S_(T1D2)) is a variable representing a signal of interestgenerated by the first signal-generating means at the second detectiontemperature, (S_(T2D2)) is a variable representing a signal of interestgenerated by the second signal-generating means at the second detectiontemperature; and the total number of variables is four;

(c) providing two additional equations each of which comprises at leastone variable selected from the group consisting of the four variables,(S_(T1D1)), (S_(T2D1)), (S_(T1D2)) and (S_(T1D2)); and

(d) obtaining solutions to at least one of the variables by the fourequations provided in the steps (b) and (c) for differentiating at leastone of the signals of interest to be assigned to at least one of the twotarget nucleic acid sequences.

According to conventional real-time PCR methods using amplificationcurves, it is common knowledge in the art that a plurality of targetnucleic add sequences cannot be differentially detected by use ofsignal-generating means providing undistinguishable identical signals.

The present invention overcomes limitations associated with the commonknowledge in the art and leads to unexpected results to detect targetnucleic acid sequences in greatly improved manner.

The present method is expressed herein as a method for differentiatingsignals of interest for each of two target nucleic add sequences in asample and alternatively may be expressed as a method for detecting atleast one of two target nucleic acid sequences in a sample or a methodfor determining the presence of at least one of two target nucleic addsequences in a sample by assigning at least one of signals of interestto at least one of the two target nucleic add sequences.

The term used herein “signal of interest” refers to a signal to bedifferentiated by the present invention. The signal finally detected maybe a sum of a plurality of signals. For example, the signal finallydetected at a detection temperature may include (i) a signal from afirst signal-generating means for a first target nucleic acid sequence(T1) and (ii) a signal from a second signal-generating means for asecond target nucleic acid sequence (T2). Each of (i) the signal and(ii) the signal is the signal of interest to be differentiated by thepresent invention. For example, the present invention aims todifferentiating (i) the signal and/or (ii) the signal from the detectedsignal. Such differentiation of the signal of interest permits to assignthe signal of interest to at least one of the two target nucleic addsequences. As a result, the differentiation of the signal of interestallows for determination whether the target nucleic add sequences arepresent or absent in the sample.

According to conventional technologies, the signals of interest to begenerated by the two signal-generating means are not differentiated foreach target nucleic acid sequence. Interestingly, the present inventionenables to differentiate the signals of interest to be generated by thetwo signal-generating means such that the signals of interest areassigned to the two target nucleic add sequences even using a singletype of detector.

Furthermore, the present invention permits to differentiate the signalsof interest by using mathematical equations in much more systematical,reliable, convenient manner.

The present invention will be described in more detail as follows:

Step (a): Incubating Samples with Signal-Generating Means and SignalDetection

The sample to be analyzed is incubated with a first signal-generatingmeans for detection of the first target nucleic add sequence (T1) and asecond signal-generating means for detection of the second targetnucleic acid sequence (T2) and then signals from the twosignal-generating means are detected at a first detection temperature(D1) and a second detection temperature (D2); wherein the signals ofinterest to be generated by the two signal-generating means are notdifferentiated for each target nucleic acid sequence by a single type ofdetector.

The present invention utilizes signal-generating means for providingsignals for target nucleic add sequences. Each of the target nucleicacid sequences is detected by a corresponding signal-generating means.

The term used herein “signal-generating means” refers to any materialused in generation of signals indicating the presence of target nucleicacid sequences, for example including oligonucleotides, labels andenzymes. Alternatively, the term used herein “signal-generating means”can be used to refer to any methods using the materials for signalgeneration.

According to an embodiment of this invention, the incubation ispreformed under conditions allowing a signal generation by thesignal-generation means. Such conditions include temperatures, saltconcentrations and pH of solutions.

Examples of the oligonucleotides serving as signal-generating meansinclude oligonucleotides to be specifically hybridized with targetnucleic add sequences (e.g., probes and primers); where probes orprimers hybridized with target nucleic acid sequences are cleaved torelease a fragment, the oligonucleotides serving as signal-generatingmeans include capture oligonucleotides to be specifically hybridizedwith the fragment; where the fragment hybridized with the captureoligonucleotide is extended to form an extended strand, theoligonucleotides serving as signal-generating means includeoligonucleotides to be specifically hybridized with the extended strand;the oligonucleotides serving as signal-generating means includeoligonucleotides to be specifically hybridized with the captureoligonucleotide; and the oligonucleotides serving as signal-generatingmeans include combinations thereof.

While a signal generation principle is the same, the signal generatingmeans comprising different sequences of oligonucleotides used may beconsidered different from each other.

The label may be linked to oligonucleotides or may be in the free form.The label may be incorporated into extended products during an extensionreaction.

Where the cleavage of oligonucleotides is used in signal generation,examples of the enzyme include 5′-nuclease and 3′-nuclease, particularlynucleic acid polymerase having 5′-nuclease activity, nucleic acidpolymerase having 3′-nuclease activity or FEN nuclease.

In the present invention, signals may be generated by using variousmaterials in various fashions.

According to an embodiment, at least one of the two signal-generatingmeans is a signal-generating means to generate a signal in a dependentmanner on the formation of a duplex.

According to an embodiment, the signal-generating means for each of thetarget nucleic add sequences are signal-generating means to generate asignal in a dependent manner on the formation of a duplex.

According to an embodiment, the duplex includes a double stranded targetnucleic add sequence.

The expression used herein “generate a signal in a dependent manner onthe formation of a duplex” in conjunction with signal-generating meansrefers to that signal to be detected is provided being dependent onassociation or dissociation of two nucleic add molecules. The expressionincludes that a signal is provided by a duplex (e.g. a detectionoligonucleotide with a label and a nucleic acid sequence) formed beingdependent on the presence of a target nucleic add sequence. In addition,the expression includes that a signal is provided by inhibition ofhybridization of a duplex (e.g. a detection oligonucleotide with a labeland a nucleic acid sequence) wherein the inhibition is caused by theformation of another duplex.

Particularly, the signal is generated by the formation of a duplexbetween a target nucleic acid sequence and a detection oligonucleotidespecifically hybridized with the target nucleic acid sequence.

The term used herein “detection oligonucleotide” is an oligonucleotidewhich is involved in generation of signal to be detected. According toan embodiment of the present invention, the detection oligonucleotideincludes an oligonucleotide which is involved in an actual signalgeneration. For example, the hybridization or non-hybridization of adetection oligonucleotide to another oligonucleotide (e.g. a targetnucleic acid sequence or an oligonucleotide comprising a nucleotidesequence complementary to the detection oligonucleotide) determines thesignal generation.

According to an embodiment of the present invention, the detectionoligonucleotide comprises at least one label.

The signal by the formation of a duplex between a target nucleic acidsequence and the detection oligonucleotide may be generated by variousmethods, including Scorpion method (Whitcombe et al, NatureBiotechnology 17:804-807 (1999)), Sunrise (or Amplifluor) method(Nazarenko et al, Nucleic Acids Research, 25(12):2516-2521 (1997), andU.S. Pat. No. 6,117,635), Lux method (U.S. Pat. No. 7,537,886), Plexormethod (Sherrill C B, et al., Journal of the American Chemical Society,126:4550-45569 (2004)), Molecular Beacon method (Tyagi et al, NatureBiotechnology v.14 Mar. 1996), HyBeacon method (French D J et al., Mol.Cell Probes, 15(6):363-374 (2001)), adjacent hybridization probe method(Bernard, P. S. et al., Anal. Biochem., 273:221 (1999)) and LNA method(U.S. Pat. No. 6,977,295).

Particularly, the signal is generated by a duplex formed in a dependentmanner on cleavage of a mediation oligonucleotide specificallyhybridized with the target nucleic add sequence.

The term used herein “mediation oligonucleotide” is an oligonucleotidewhich mediates production of a duplex not containing a target nucleicadd sequence.

According to an embodiment of the present invention, the cleavage of themediation oligonucleotide per se does not generate signal and a fragmentformed by the cleavage is involved in successive reactions for signalgeneration following hybridization and cleavage of the mediationoligonucleotide.

According to an embodiment, the hybridization or cleavage of themediation oligonucleotide per se does not generate signal.

According to an embodiment of the present invention, the mediationoligonucleotide includes an oligonucleotide which is hybridized with atarget nucleic acid sequence and cleaved to release a fragment, leadingto mediate the production of a duplex. Particularly, the fragmentmediates a production of a duplex by an extension of the fragment on acapture oligonucleotide.

According to an embodiment of the present invention, the mediationoligonucleotide comprises (i) a 3′-targeting portion comprising ahybridizing nucleotide sequence complementary to the target nucleic addsequence and (ii) a 5′-tagging portion comprising a nucleotide sequencenon-complementary to the target nucleic acid sequence.

According to an embodiment of the present invention, the cleavage of amediation oligonucleotide release a fragment and the fragment isspecifically hybridized with a capture oligonucleotide and extended onthe capture oligonucleotide.

According to an embodiment of the present invention, a mediationoligonucleotide hybridized with target nucleic add sequences is cleavedto release a fragment and the fragment is specifically hybridized with acapture oligonucleotide and the fragment is extended to form an extendedstrand, resulting in formation of a extended duplex between the extendedstand and the capture oligonucleotide providing a signal indicating thepresence of the target nucleic acid sequence.

According to an embodiment of the present invention, where a thirdoligonucleotide comprising a hybridizing nucleotide sequencecomplementary to the extended strand is used, the hybridization of thethird oligonucleotide and the extended strand forms other type of aduplex providing a signal indicating the presence of the target nucleicadd sequence.

According to an embodiment of the present invention, where a thirdoligonucleotide comprising a hybridizing nucleotide sequencecomplementary to the capture oligonucleotide is used, the formation of aduplex between the third oligonucleotide and the capture oligonucleotideis inhibited by the formation of the duplex between the extended strandand the capturing oligonucleotide, leading to provide a signalindicating the presence of the target nucleic acid sequence.

According to an embodiment of the present invention, the fragment, theextended strand, the capture oligonucleotide, the third oligonucleotideor combination of them can work as the detection oligonucleotide.

The signal by the duplex formed in a dependent manner on cleavage of themediation oligonucleotide may be generated by various methods, includingPTOCE (PTO cleavage and extension) method (WO 2012/096523), PCE-SH (PTOCleavage and Extension-Dependent Signaling OligonucleotideHybridization) method (WO 2013/115442) and PCE-NH (PTO Cleavage andExtension-Dependent Non-Hybridization) method (PCT/KR2013/012312).

With referring to terms disclosed in the above references, thecorresponding examples of the oligonucleotides are as follows: amediation oligonucleotide is corresponding to a PTO (Probing and TaggingOligonucleotide), a capture oligonucleotide to a CTO (Capturing andTemplating Oligonucleotide), and a third oligonucleotide to SO(Signaling Oligonucleotide) or HO (Hybridization Oligonucleotide),respectively. SO, HO, CTO, extended strand or their combination can takea role as a detection oligonucleotide.

The signal by the duplex formed in a dependent manner on cleavage of themediation oligonucleotide includes the signal provided by inhibition ofthe formation of other duplex by the duplex formed in a dependent manneron cleavage of the mediation oligonucleotide (e.g. PCE-NH).

For example, where the signal by the duplex formed in a dependent manneron cleavage of the mediation oligonucleotide is generated by PTOCEmethod, the signal-generating means comprises an upstreamoligonucleotide and a PTO (Probing and Tagging Oligonucleotide)comprising a hybridizing nucleotide sequence complementary to the targetnucleic add sequence, a CTO (Capturing and Templating Oligonucleotide),suitable label and a template-dependent nucleic acid polymerase having5′ nuclease activity. The PTO comprises (i) a 3′-targeting portioncomprising a hybridizing nucleotide sequence complementary to the targetnucleic acid sequence and (ii) a 5′-tagging portion comprising anucleotide sequence non-complementary to the target nucleic addsequence. The CTO comprises in a 3′ to 5′ direction (i) a capturingportion comprising a nucleotide sequence complementary to the 5′-taggingportion or a part of the 5′-tagging portion of the PTO and (ii) atemplating portion comprising a nucleotide sequence non-complementary tothe 5′-tagging portion and the 3′-targeting portion of the PTO.

The particular example of the signal generation by PTOCE methodcomprises the steps of:

(a) hybridizing the target nucleic acid sequence with the upstreamoligonucleotide and the PTO; (b) contacting the resultant of the step(a) to an enzyme having a 5′ nuclease activity under conditions forcleavage of the PTO; wherein the upstream oligonucleotide or itsextended strand induces cleavage of the PTO by the enzyme having the 5′nuclease activity such that the cleavage releases a fragment comprisingthe 5′-tagging portion or a part of the 5′-tagging portion of the PTO;(c) hybridizing the fragment released from the PTO with the CTO; whereinthe fragment released from the PTO is hybridized with the capturingportion of the CTO; and (d) performing an extension reaction using theresultant of the step (c) and a template-dependent nucleic acidpolymerase; wherein the fragment hybridized with the capturing portionof the CTO is extended and an extended duplex is formed; wherein theextended duplex has a T_(m) value adjustable by (i) a sequence and/orlength of the fragment, (ii) a sequence and/or length of the CTO or(iii) the sequence and/or length of the fragment and the sequence and/orlength of the CTO; wherein the extended duplex provides a target signalby (i) at least one label linked to the fragment and/or the CTO, (ii) alabel incorporated into the extended duplex during the extensionreaction, (iii) a label incorporated into the extended duplex during theextension reaction and a label linked to the fragment and/or the CTO, or(iv) an intercalating label; and (e) detecting the extended duplex bymeasuring the target signal at a predetermined temperature that theextended duplex maintains its double-stranded form, whereby the presenceof the extended duplex indicates the presence of the target nucleic addsequence. In this case, the method further comprises repeating all orsome of the steps (a)-(e) with denaturation between repeating cycles.

In the phrase “denaturation between repeating cycles”, the term“denaturation” means to separate a double-stranded nucleic acid moleculeto a single-stranded nucleic acid molecule.

In the step (a) of PTOCE method, a primer set for amplification of thetarget nucleic add sequence may be used instead of the upstreamoligonucleotide. In this case, the method further comprises repeatingall or some of the steps (a)-(e) with denaturation between repeatingcycles.

The PTOCE method can be classified as a process in which the PTOfragment hybridized with the CTO is extended to form an extended strandand the extended strand is then detected. The PTOCE method ischaracterized that the formation of the extended strand is detected byusing the duplex between the extended strand and the CTO.

There is another approach to detect the formation of the extendedstrand. For example, the formation of the extended strand may bedetected by using an oligonucleotide specifically hybridized with theextended strand (e.g., PCE-SH method). In this method, the signal may beprovided from (i) a label linked to the oligonucleotide specificallyhybridized with the extended strand, (ii) a label linked to theoligonucleotide specifically hybridized with the extended strand and alabel linked to the PTO fragment, (iii) a label linked to theoligonucleotide specifically hybridized with the extended strand and alabel incorporated into the extended strand during the extensionreaction, or (iv) a label linked to the oligonucleotide specificallyhybridized with the extended strand and an intercalating dye.Alternatively, the signal may be provided from (i) a label linked to theextended strand or (ii) an intercalating dye.

Alternatively, the detection of the formation of the extended strand isperformed by another method in which inhibition of the hybridizationbetween the CTO and an oligonucleotide being specifically hybridizablewith the CTO is detected (e.g. PCE-NH method). Such inhibition isconsidered to be indicative of the presence of a target nucleic addsequence. The signal may be provided from (i) a label linked to theoligonucleotide being hybridizable with the CTO, (ii) a label linked tothe CTO, (iii) a label linked to the oligonucleotide being hybridizablewith the CTO and a label linked to the CTO, or (iv) an intercalatinglabel.

According to an embodiment, the oligonucleotide being specificallyhybridizable with the CTO has an overlapping sequence with the PTOfragment.

According to an embodiment, the detection oligonucleotide includes theoligonucleotide being specifically hybridizable with the extended strand(e.g., PCE-SH method) and oligonucleotide being specificallyhybridizable with the CTO (e.g. PCE-NH method). According to anembodiment, the detection oligonucleotide includes the extended strandproduced during a reaction or CTO.

The PTOCE-based methods commonly involve the formation of the extendedstrand depending on the presence of a target nucleic acid sequence. Theterm “PTOCE-based method” is used herein to intend to encompass variousmethods for providing signals comprising the formation of an extendedstrand through cleavage and extension of PTO.

The example of signal generation by the PTOCE-based methods comprisesthe steps of: (a) hybridizing the target nucleic acid sequence with theupstream oligonucleotide and the PTO; (b) contacting the resultant ofthe step (a) to an enzyme having a 5′ nuclease activity under conditionsfor cleavage of the PTO; wherein the upstream oligonucleotide or itsextended strand induces cleavage of the PTO by the enzyme having the 5′nuclease activity such that the cleavage releases a fragment comprisingthe 5′-tagging portion or a part of the 5′-tagging portion of the PTO;(c) hybridizing the fragment released from the PTO with the CTO; whereinthe fragment released from the PTO is hybridized with the capturingportion of the CTO; (d) performing an extension reaction using theresultant of the step (c) and a template-dependent nucleic addpolymerase; wherein the fragment hybridized with the capturing portionof the CTO is extended to form an extended strand; and (e) detecting theformation of the extended strand by detecting signal generated dependenton the presence of the extended strand. In the step (a), a primer setfor amplification of the target nucleic acid sequence may be usedinstead of the upstream oligonucleotide. In this case, the methodfurther comprises repeating all or some of the steps (a)-(e) withdenaturation between repeating cycles.

According to an embodiment, the signal generated by the formation of aduplex includes signals induced by hybridization of the duplex (e.g.,hybridization of the duplex per se, or hybridization of a thirdoligonucleotide) or by inhibition of hybridization of a thirdoligonucleotide due to the formation of a duplex.

According to an embodiment, the signal-generating means for at least oneof the target nucleic acid sequences is a signal-generating means byformation of a duplex in a dependent manner on cleavage of a mediationoligonucleotide specifically hybridized with the target nucleic addsequence.

According to an embodiment, the signal-generating means for each of thetarget nucleic acid sequences are a signal-generating means by formationof a duplex in a dependent manner on cleavage of a mediationoligonucleotide specifically hybridized with the target nucleic addsequence.

According to an embodiment, at least one of the two signal-generatingmeans is a signal-generating means to generate a signal in a dependentmanner on cleavage of a detection oligonucleotide.

According to an embodiment, the signal-generating means for each of thetarget nucleic acid sequences are signal-generating means to generate asignal in a dependent manner on cleavage of a detection oligonucleotide.

Particularly, the signal is generated by hybridization of the detectionoligonucleotide with a target nucleic add sequence and then cleavage ofthe detection oligonucleotide.

The signal by hybridization of the detection oligonucleotide with atarget nucleic add sequence and then cleavage of the detectionoligonucleotide may be generated by various methods, including TaqManprobe method (U.S. Pat. Nos. 5,210,015 and 5,538,848).

Where the signal is generated by TaqMan probe method, thesignal-generating means includes a primer set for amplification of atarget nucleic acid sequence, TaqMan probe having a suitable label(e.g., interactive dual label) and nucleic acid polymerase having5′-nuclease activity. The TaqMan probe hybridized with a target nucleicacid sequence is cleaved during target amplification and generatessignal indicating the presence of the target nucleic acid sequence.

The particular example generating signal by TaqMan probe methodcomprises the step of: (a) hybridizing the primer set and TaqMan probehaving a suitable label (e.g., interactive dual label) with the targetnucleic acid sequence; (b) amplifying the target nucleic acid sequenceby using the resultant of the step (a) and nucleic acid polymerasehaving 5′-nuclease activity, wherein the TaqMan probe is cleaved torelease the label; and (c) detecting a signal generation from thereleased label.

Particularly, the signal is generated by cleavage of the detectionoligonucleotide in a dependent manner on cleavage of a mediationoligonucleotide specifically hybridized with the target nucleic addsequence.

According to an embodiment, the signal-generating means for at least oneof the target nucleic acid sequences is a signal-generating means togenerate a signal by cleavage of the detection oligonucleotide in adependent manner on cleavage of a mediation oligonucleotide specificallyhybridized with the target nucleic add sequence.

According to an embodiment, the signal-generating means for each of thetarget nucleic acid sequences are signal-generating means to generate togenerate a signal by cleavage of the detection oligonucleotide in adependent manner on cleavage of a mediation oligonucleotide specificallyhybridized with the target nucleic acid sequence.

According to an embodiment of the present invention, where a mediationoligonucleotide hybridized with target nucleic add sequences is cleavedto release a fragment, the fragment is specifically hybridized with adetection oligonucleotide and the fragment induces the cleavage of thedetection oligonucleotide.

According to an embodiment of the present invention, where a mediationoligonucleotide hybridized with target nucleic add sequences is cleavedto release a fragment, the fragment is extended to cleave a detectionoligonucleotide comprising a hybridizing nucleotide sequencecomplementary to the capture oligonucleotide.

The signal by cleavage of the detection oligonucleotide in a dependentmanner on cleavage of the mediation oligonucleotide may be generated byvarious methods, Including Invader assay (U.S. Pat. No. 5,691,142), PCEC(PTO Cleavage and Extension-Dependent Cleavage) method (WO 2012/134195)and a method described in U.S. Pat. No. 7,309,573. In particular, themethod described in U.S. Pat. No. 7,309,573 may be considered as one ofPTOCE-based methods using signal generation by cleavage, and in themethod, the formation of the extended strand may be detected bydetecting cleavage of an oligonucleotide specifically hybridized withthe CTO by the formation of the extended strand. Invader assay forms afragment by cleavage of a mediation oligonucleotide and inducessuccessive cleavage reactions with no extension of the fragment.

According to an embodiment of the present invention, where the signal isgenerated in a dependent manner on cleavage of a detectionoligonucleotide, the cleavage of the detection oligonucleotide inducessignal changes or releases a labeled fragment to be detected.

Where a signal-generating means generates a signal by cleavage of adetection oligonucleotide as well as by the formation of a duplex, thesignal-generating means may be considered as a signal generating meansproviding signal by cleavage, so long as it is used to generate signalby cleavage.

Where the signal is generated by cleavage of the detectionoligonucleotide, a released label by the cleavage may be detected at anytemperatures.

According to the embodiment of this invention, the signal-generatingmeans for the target nucleic acid sequences are combination of asignal-generating means by cleavage of a detection oligonucleotide, anda signal-generating means by the formation of a duplex.

According to an embodiment, the detection oligonucleotide comprises atleast one label.

According to an embodiment of the present invention, the detectionoligonucleotide may be composed of at least one oligonucleotide.According to an embodiment of the present invention, where the detectionoligonucleotide is composed of a plurality of oligonucleotides, it mayhave a label in various manners. For instance, one oligonucleotide amonga plurality of oligonucleotides may have at least one label, a pluralityof oligonucleotides all may have at least one label, or one portion ofoligonucleotides may have at least one label and the other portion maynot have a label.

The signals generated by the two signal-generating means are notdifferentiated by a single type of detector. The term “signals notdifferentiated by a single type of detector” means that signals are notdifferentiated from each other by a single type of detector due to theiridentical or substantially identical signal properties (e.g., opticalproperties, emission wavelength and electrical signal). For example,where the same label (e.g., FAM) is used for two target nucleic acidsequences and a single type of detector for detection of emissionwavelength from FAM is used, signals are not differentially detected.

The term used herein “a single type of signal” means signals providingidentical or substantially identical signal properties (e.g., opticalproperties, emission wavelength and electrical signal). For example, FAMand CAL Fluor 610 provide different types of signals.

The term used herein “a single type of detector” means a detection meansfor a singly type of signal. In a detector comprising several channels(e.g., photodiodes) for several different types of signals, each channel(e.g., a photodiode) corresponds to “a single type of detector”.

According to an embodiment of this invention, the two signal-generatingmeans comprise an identical label and signals from the label are notdifferentiated by the single type of detector.

The label useful in the present invention includes various labels knownin the art. For example, the label useful in the present inventionincludes a single label, an interactive dual label, an intercalating dyeand an incorporating label.

The single label includes, for example, a fluorescent label, aluminescent label, a chemiluminescent label, an electrochemical labeland a metal label. According to an embodiment, the single label providesa different signal (e.g., different signal intensities) depending on itspresence on a double strand or single strand. According to anembodiment, the single label is a fluorescent label. The preferabletypes and binding sites of single fluorescent labels used in thisinvention are disclosed U.S. Pat. Nos. 7,537,886 and 7,348,141, theteachings of which are incorporated herein by reference in their entity.For example, the single fluorescent label includes JOE, FAM, TAMRA, ROXand fluorescein-based label. The single label may be linked tooligonucleotides by various methods. For instance, the label is linkedto probes through a spacer containing carbon atoms (e.g., 3-carbonspacer, 6-carbon spacer or 12-carbon spacer).

As a representative of the interactive label system, the FRET(fluorescence resonance energy transfer) label system includes afluorescent reporter molecule (donor molecule) and a quencher molecule(acceptor molecule). In FRET, the energy donor is fluorescent, but theenergy acceptor may be fluorescent or non-fluorescent. In another formof interactive label systems, the energy donor is non-fluorescent, e.g.,a chromophore, and the energy acceptor is fluorescent. In yet anotherform of interactive label systems, the energy donor is luminescent, e.g.bioluminescent, chemiluminescent, electrochemiluminescent, and theacceptor is fluorescent. The interactive label system includes a duallabel based on “on contact-mediated quenching” (Salvatore et al.,Nucleic Adds Research, 2002 (30) no. 21 e122 and Johansson et al., J.AM. CHEM. SOC 2002 (124) pp 6950-6956). The interactive label systemincludes any label system in which signal change is induced byinteraction between at least two molecules (e.g. dye).

The reporter molecule and the quencher molecule useful in the presentinvention may include any molecules known in the art. Examples of thoseare: Cy2™ (506), YO-PRO™-1 (509), YOYO™-1 (509), Calcein (517), FITC(518), FluorX™ (519), Alexa™ (520), Rhodamine 110 (520), Oregon Green™500 (522), Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™(527), Rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™(533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil(565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3™(570), Alexa™ 546 (570), TRITC (572), Magnesium Orange™ (575),Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange™(576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red™(590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594(615), Texas Red (615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631),R-phycocyanin (642), C-Phycocyanin (648), TO-PRO™-3 (660), TOTO3 (660),DiD DilC(5) (665), Cy5™ (670), Thiadicarbocyanine (671), Cy5.5 (694),HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544),CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610(610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520),Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670(705) and Quasar 705 (610). The numeric in parenthesis is a maximumemission wavelength in nanometer. Preferably, the reporter molecule andthe quencher molecule include JOE, FAM, TAMRA, ROX and fluorescein-basedlabel.

Suitable fluorescence molecule and suitable pairs of reporter-quencherare disclosed in a variety of publications as follows: Pesce et al.,editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971);White et al., Fluorescence Analysis: A Practical Approach (MarcelDekker, New York, 1970); Berlman, Handbook of Fluorescence Spectra ofAromatic Molecules, 2^(nd) Edition (Academic Press, New York, 1971);Griffiths, Color AND Constitution of Organic Molecules (Academic Press,New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford,1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals(Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence andPhosphorescence (Interscience Publishers, New York, 1949); Haugland, R.P., Handbook of Fluorescent Probes and Research Chemicals, 6^(th)Edition (Molecular Probes, Eugene, Oreg., 1996) U.S. Pat. Nos. 3,996,345and 4,351,760.

It is noteworthy that a non-fluorescent quencher molecule (e.g. blackquencher or dark quencher) capable of quenching a fluorescence of a widerange of wavelengths or a specific wavelength may be used in the presentinvention.

In the signaling system comprising the reporter and quencher molecules,the reporter encompasses a donor of FRET and the quencher encompassesthe other partner (acceptor) of FRET. For example, a fluorescein dye isused as the reporter and a rhodamine dye as the quencher.

The interactive dual label may be linked to one strand of a duplex.Where the strand containing the interactive dual label leaves in asingle stranded state, it forms a hairpin or random coil structure toinduce quenching between the interactive dual label. Where the strandforms a duplex, the quenching is relieved. Alternatively, where theinteractive dual label is linked to nucleotides adjacently positioned onthe strand, the quenching between the interactive dual label occurs.Where the strand forms a duplex and then is cleaved, the quenchingbecomes relieved.

Each of the interactive dual label may be linked to each of two strandsof the duplex. The formation of the duplex induces quenching anddenaturation of the duplex induces unquenching. Alternatively, where oneof the two stands is cleaved, the unquenching may be induced.

Exemplified intercalating dyes useful in this invention include SYBR™Green I, PO-PRO™-1, BO-PRO™-1, SYTO™43, SYTO™44, SYTO™45, SYTOX™Blue,POPO™-1, POPO™-3, BOBO™-1, BOBO™-3, LO-PRO™-1, JO-PRO™-1, YO-PRO™1,TO-PRO™1, SYTO™11, SYTO™13, SYTO™15, SYTO™16, SYTO™20, SYTO™23, TOTO™-3,YOYO™3, GelStar™ and thiazole orange. The intercalating dyes intercalatespecifically into double-stranded nucleic add molecules to generatesignals.

The incorporating label may be used in a process to generate signals byincorporating a label during primer extension (e.g., Plexor method,Sherrill C B, et al., Journal of the American Chemical Society,126:4550-45569 (2004)). The incorporating label may be also used in asignal generation by a duplex formed in a dependent manner on cleavageof a mediation oligonucleotide hybridized with the target nucleic addsequence.

The incorporating label may be generally linked to nucleotides. Thenucleotide having a non-natural base may be also used.

The term used herein “non-natural base” refers to derivatives of naturalbases such as adenine (A), guanine (G), thymine (T), cytosine (C) anduracil (U), which are capable of forming hydrogen-bonding base pairs.The term used herein “non-natural base” includes bases having differentbase pairing patterns from natural bases as mother compounds, asdescribed, for example, in U.S. Pat. Nos. 5,432,272, 5,965,364,6,001,983, and 6,037,120. The base pairing between non-natural basesinvolves two or three hydrogen bonds as natural bases. The base pairingbetween non-natural bases is also formed in a specific manner. Specificexamples of non-natural bases include the following bases in base paircombinations: iso-C/iso-G, iso-dC/iso-dG, K/X, H/J, and M/N (see U.S.Pat. No. 7,422,850).

Where the signal is generated by the PTOCE method, a nucleotideincorporated during the extension reaction may have a first non-naturalbase and the CTO may have a nucleotide having a second non-natural basewith a specific binding affinity to the first non-natural base.

The term used herein “target nucleic add”, “target nucleic acidsequence” or “target sequence” refers to a nucleic acid sequence ofinterest for detection or quantification. The target nucleic addsequence comprises a sequence in a single strand as well as in a doublestrand. The target nucleic acid sequence comprises a sequence initiallypresent in a nucleic add sample as well as a sequence newly generated inreactions.

The target nucleic add sequence may include any DNA (gDNA and cDNA), RNAmolecules their hybrids (chimera nucleic add). The sequence may be ineither a double-stranded or single-stranded form. Where the nucleic addas starting material is double-stranded, it is preferred to render thetwo strands into a single-stranded or partially single-stranded form.Methods known to separate strands includes, but not limited to, heating,alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g.,helicase action), and binding proteins. For instance, strand separationcan be achieved by heating at temperature ranging from 80° C. to 105° C.General methods for accomplishing this treatment are provided by JosephSambrook, et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

Where a mRNA is employed as starting material, a reverse transcriptionstep is necessary prior to performing annealing step, details of whichare found in Joseph Sambrook, et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and Noonan, K. F. et al., Nucleic Adds Res. 16:10366 (1988)).For reverse transcription, an oligonucleotide dT primer hybridizable topoly A tall of mRNA, random primers or target-specific primers may beused.

The target nucleic acid sequence includes any naturally occurringprokaryotic, eukaryotic (for example, protozoans and parasites, fungi,yeast, higher plants, lower and higher animals, including mammals andhumans), viral (for example, Herpes viruses, HIV, influenza virus,Epstein-Barr virus, hepatitis virus, polio virus, etc.), or viroidnucleic add. The nucleic add molecule can also be any nucleic acidmolecule which has been or can be recombinantly produced or chemicallysynthesized. Thus, the nucleic add sequence may or may not be found innature. The target nucleic acid sequence may include known or unknownsequences.

The term used herein “sample” refers to any cell, tissue, or fluid froma biological source, or any other medium that can advantageously beevaluated according to this invention, including virus, bacteria,tissue, cell, blood, serum, plasma, lymph, milk, urine, faeces, ocularfluid, saliva, semen, brain extracts, spinal cord fluid (SCF), appendix,spleen and tonsillar tissue extracts, amniotic fluid, ascitic fluid andnon-biological samples (e.g., food and water). In addition, the sampleincludes natural-occurring nucleic add molecules isolated frombiological sources and synthetic nucleic acid molecules.

Signals include various signal characteristics from the signaldetection, e.g., signal intensity [e.g., RFU (relative fluorescenceunit) value or in the case of performing amplification, RFU values at acertain cycle, at selected cycles or at end-point], signal change shape(or pattern) or C_(t) value, or values obtained by mathematicallyprocessing the characteristics.

According to an embodiment, the term “signal” with conjunction withreference value or sample analysis includes not only signals per seobtained at detection temperatures but also a modified signal providedby mathematically processing the signals.

According to an embodiment of this invention, when an amplificationcurve is obtained by real-time PCR, various signal values (orcharacteristics) from the amplification curve may be selected used fordifferentiation of signal values (or characteristics) at each detectiontemperature and detection of the target nucleic acid sequence(intensity, C_(t) value or amplification curve data).

According to an embodiment of this invention, the step (a) is performedin a signal amplification process concomitantly with a nucleic acidamplification. According to an embodiment of this invention, wherein thestep (a) is performed in a signal amplification process without anucleic add amplification.

In the present invention, the signal generated by signal-generatingmeans may be amplified simultaneously with target amplification.Alternatively, the signal may be amplified with no target amplification.

According to an embodiment of this invention, the signal generation isperformed in a process involving signal amplification together withtarget amplification.

According to an embodiment of this invention, the target amplificationis performed in accordance with PCR (polymerase chain reaction). PCR iswidely employed for target amplification in the art, including cycles ofdenaturation of a target sequence, annealing (hybridization) between thetarget sequence and primers and primer extension (Mullis et al. U.S.Pat. Nos. 4,683,195, 4,683,202 and 4,800,159; Saiki et al., (1985)Science 230, 1350-1354). The signal may be amplified by applying thesignal generation methods described above (e.g., TaqMan method andPTOCE-based methods) to the PCR process. According to an embodiment, thepresent invention provides signals by real-time PCR method. According toan embodiment, the amplification of the target nucleic acid sequence isperformed by PCR (polymerase chain reaction), LCR (ligase chainreaction, see Wiedmann M, et al., “Ligase chain reaction (LCR)—overviewand applications.” PCR Methods and Applications 1994 February;3(4):S51-64), GLCR (gap filling LCR, see WO 90/01069, EP 439182 and WO93/00447), Q-beta (Q-beta replicase amplification, see Cahill P, et al.,Clin Chem., 37(9):1482-5 (1991), U.S. Pat. No. 5,556,751), SDA (stranddisplacement amplification, see G T Walker et al., Nucleic Adds Res.20(7):16911696 (1992), EP 497272), NASBA (nucleic add sequence-basedamplification, see Compton, J. Nature 350(6313):912 (1991)), TMA(Transcription-Mediated Amplification, see Hofmann W P et al., J ClinVirol. 32(4):289-93 (2005); U.S. Pat. No. 5,888,779).) or RCA (RollingCircle Amplification, see Hutchison C. A. et al., Proc. Natl Acad. Sci.USA. 102:1733217336 (2005)).

The amplification methods described above may amplify target sequencesthrough repeating a series of reactions with or without changingtemperatures. The unit of amplification comprising the repetition of aseries of reactions is expressed as a “cycle”. The unit of cycles may beexpressed as the number of the repetition or time being dependent onamplification methods.

For example, the detection of signals may be performed at each cycle ofamplification, selected several cycles or end-point of reactions.According to an embodiment, where signals are detected at at least twocycles, the detection of signal in an individual cycle may be performedat all detection temperatures or some selected detection temperatures.According to an embodiment of this invention, the detection is performedat the relatively high detection temperature in odd numbered cycles andat the relatively high detection temperature in even numbered cycles.

According to an embodiment of this invention, the incubation ispreformed in the conditions allowing target amplification well as signalgeneration by the signal-generation means.

The amplification of the target nucleic add sequence is accomplished bytarget amplification means including a primer set for amplification andnucleic acid polymerase.

According to an embodiment of the present invention, a nucleic acidpolymerase having a nuclease activity (e.g. 5′ nuclease activity or 3′nuclease activity) may be used. According to an embodiment of thepresent invention, a nucleic acid polymerase having a no nucleaseactivity may be used.

The nucleic acid polymerase useful in the present invention is athermostable DNA polymerase obtained from a variety of bacterialspecies, including Thermus aquaticus (Taq), Thermus thermophilus (Tth),Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermusantranikianii, Thermus caldophilus, Thermus chliarophilus, Thermusflavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermusruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermusspecies Z05, Thermus species sps 17, Thermus thermophilus, Thermotogamaritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcuslitoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotogamaritima, Thermotoga neapolitana, Thermosipho africanus, Pyrococcuswoesei, Pyrococcus horikoshii, Pyrococcus abyssi, Pyrodictium occultum,Aquifex pyrophilus and Aquifex aeolieus. Particularly, the thermostableDNA polymerase is Taq polymerase.

According to an embodiment of the present invention, the amplificationof the target nucleic add sequence is accomplished by an asymmetric PCR.The ratio of primers may be selected in consideration of cleavage orhybridization of downstream oligonucleotides.

According to an embodiment of this invention, the step (a) is performedin a signal amplification process without a nucleic add amplification.

Where the signal is generated by methods including cleavage of anoligonucleotide, the signal may be amplified with no targetamplification. For example, the step (a) may be performed withamplification of signals but with no amplification of target sequencesin accordance with CPT method (Duck P, et al., Biotechniques, 9:142-148(1990)), Invader assay (U.S. Pat. Nos. 6,358,691 and 6,194,149),PTOCE-based methods (e.g., PCE-SH method, PCE-NH method and PCEC method)or CER method (WO 2011/037306).

The signal amplification methods described above may amplify signalsthrough repeating a series of reactions with or without changingtemperatures. The unit of signal amplification comprising the repetitionof a series of reactions is expressed as a “cycle”. The unit of cyclesmay be expressed as the number of the repetition or time being dependenton amplification methods.

For example, the generation and detection of signals may be performed ateach cycle of amplification, selected several cycles or end-point ofreactions.

During or after the incubation (reaction) of the sample with twosignal-generating means to provide signals, the signals are detected byusing a detector such as a single type of detector.

The present invention may be performed by using any type ofsignal-generating means in view of detection temperatures for detectionof a plurality of target nucleic add sequence. The signal-generatingmeans may be designed to provide signals at both of the two detectiontemperatures or at only one of the two detection temperatures in thepresence of a target nucleic acid sequence. Where the signal-generatingmeans is designed to provide signals at only one of the two detectiontemperatures, it is advantageous that it provide signals only at lowerdetection temperature.

According to an embodiment, each of two target nucleic acid sequence isdetected by a signal-generating means capable of generating signals atall detection temperatures.

According to an embodiment, 1^(st) target nucleic add sequence among twotarget nucleic add sequences is detected by a signal-generating meanscapable of generating signals at one detection temperatures and a 2^(nd)target nucleic acid sequence of two target nucleic add sequences isdetected by a signal-generating means capable of generating signals attwo detection temperatures.

According to an embodiment, the detection temperatures are predeterminedin considering a temperature range to allow signal generation by thesignal-generating means.

The present invention uses that there is a certain temperature range toallow signal generation in a dependent manner on signal-generatingmeans.

For example, when a signal-generating means generates a signal uponhybridization (or association) between two nucleic add molecules and donot generate a signal upon non-hybridization (or dissociation) betweenthem, a signal is generated at temperatures allowing hybridizationbetween two nucleic acid molecules, however, no signal is generated attemperatures failing to hybridize between two nucleic acid molecules. Assuch, there is a certain temperature range to allow signal generation(I.e., signal detection) and other temperature range not to allow signalgeneration. The temperature ranges are affected by the T_(m) value ofthe hybrid of the two nucleic acid molecules employed in thesignal-generation means.

Where the signal generation method using a released fragment with alabel after cleavage is employed, the signal may be theoreticallydetected at any temperature (e.g., 30-99° C.).

A detection temperature is selected from the temperature range to allowsignal generation by the signal generation mean.

According to an embodiment, the signal-generating means for detecting atarget nucleic add sequence may be signal-generating means to providedifferent signals (e.g. signal intensity) or different signal changepattern (e.g. change extent of signal intensities) from each other atthe two detection temperatures.

According to an embodiment, when the signal-generating means to generatesignals in a dependent manner on cleavage of a detection oligonucleotide(e.g. TaqMan probe method) is employed, signals from thesignal-generating means may be different depending on detectiontemperatures in the sense that signal generation by hybridization ofdetection oligonucleotides with target nucleic add sequences and/orsignal generation from dyes may be affected by temperatures. Forinstance, a probe labeled with a fluorescent reporter molecule and/or aquencher molecule may generate different signals depending on detectiontemperatures in which the probe may generate higher signals at therelatively low detection temperature than those at the relatively highdetection temperature.

According to an embodiment, signal-generating means for the two targetnucleic acid sequences are firstly constructed and then detectiontemperatures for the two target nucleic add sequences are allocated,followed by performing the step (a).

According to an embodiment of this invention, when the signal-generatingmeans generates a signal in a dependent manner on the formation of aduplex, the detection temperature is selected based on a T_(m) value ofthe duplex.

According to an embodiment of this invention, when the signal-generatingmeans generates a signal in a dependent manner on the formation of aduplex, the detection temperature is controllable by adjusting a T_(m)value of the duplex.

For example, where the signal is generated by a detectionoligonucleotide specifically hybridized with the target nucleic addsequence (e.g., Lux probe, Molecular Beacon probe, HyBeacon probe andadjacent hybridization probe), the detection of the signal issuccessfully done at the predetermined temperature by adjusting theT_(m) value of the oligonucleotide. Where Scorpion primer is used, thedetection of the signal is successfully done at the predeterminedtemperature by adjusting the T_(m) value of a portion to be hybridizedwith extended strand.

Where the signal is generated by the duplex formed dependent on thepresence of the target nucleic acid sequence, the detection of thesignal is successfully done at the predetermined temperature byadjusting the T_(m) value of the duplex. For example, where the signalis generated by the PTOCE method, the detection of the signal issuccessfully done at the predetermined temperature by adjusting theT_(m) value of the extended duplex formed by the extension of the PTOfragment on the CTO.

The PTOCE-based methods have advantages to readily adjust T_(m) valuesof the duplex or a third hybrid whose hybridization is affected by theduplex.

According to an embodiment of this invention, when the signal-generatingmeans generates a signal in a dependent manner on cleavage of adetection oligonucleotide, the detection temperature is arbitrarilyselected. In other words, any temperature can be selected so long as thesignal generated by cleavage of a detection oligonucleotide may bedetected. As described above, where the signal is generated beingdependent manner on cleavage of the detection oligonucleotide, the labelreleased by the cleavage may be detected at various temperatures.

Both of signal-generating means for the two target nucleic acidsequences may be designed to provide signals at both of the twodetection temperatures. According to an embodiment, TaqMan probe method,PTOCE-based methods or their combination may be selected assignal-generating means to provide signals at both of the two detectiontemperatures.

Alternatively, the signal-generating means for one of the two targetnucleic add sequences may be designed to provide signals at both of thetwo detection temperatures and the signal-generating means for the othertarget nucleic acid sequence may be designed to provide a signal at onedetection temperature (particularly, at relatively lower detectiontemperature). According to an embodiment, TaqMan probe method orPTOCE-based methods may be selected to provide signals at both of thetwo detection temperatures and PTOCE-based methods may be selected toprovide a signal at one detection temperature.

The detector used in the present invention includes any means capable ofdetecting signals. For example, where fluorescent signals are used,photodiodes suitable in detection of the fluorescent signals may beemployed as detectors. The detection using a single type of detectormeans that the detection is performed by using a detector capable ofdetecting a single type of signal or using each channel (i.e.,photodiode) of a detector carrying several channels (i.e., photodiodes).

According to an embodiment, the generation of signals includes “signalgeneration or extinguishment” and “signal increase or decrease” fromlabels.

Step (b): Establishing Equations Comprising Variables RepresentingSignals of Interest

Provided are the following two equations each of which comprisesvariables representing the signals of interest generated at eachdetection temperature for the two target nucleic add sequences:S _(T1D1) +S _(T2D1) =S _(D1)  (I)S _(T1D2) +S _(T2D2) =S _(D2)  (II)

wherein (S_(D1)) is a signal detected at the first detectiontemperature, (S_(D2)) is a signal detected at the second detectiontemperature; (S_(T1D1)) is a variable representing a signal of interestgenerated by the first signal-generating means at the first detectiontemperature, (S_(T2D1)) is a variable representing a signal of interestgenerated by the second signal-generating means at the first detectiontemperature, (S_(T1D2)) is a variable representing a signal of interestgenerated by the first signal-generating means at the second detectiontemperature, (S_(T2D2)) is a variable representing a signal of interestgenerated by the second signal-generating means at the second detectiontemperature; and the total number of variables is four.

(S_(D1)), the signal detected at the first detection temperature (D1)consists of (S_(T1D1)), the signal of interest generated by the firstsignal-generating means at the first detection temperature (D1) and(S_(T2D1)), the signal of interest generated by the secondsignal-generating means at the first detection temperature (D1).(S_(D2)), the signal detected at the second detection temperature (D2)consists of (S_(T1D2)), the signal of interest generated by the firstsignal-generating means at the second detection temperature (D2) and(S_(T2D2)), the signal of interest generated by the secondsignal-generating means at the second detection temperature (D2).

(S_(D1)) and (S_(D2)) may be a measured signal by detectors, I.e., aconstant value. (S_(T1D1)), (S_(T2D1)), (S_(T1D2)) and (S_(T2D2)) areunknown variables to be obtained. The present invention is drawn tomethods for obtaining solutions to at least one of the variables(S_(T1D1)), (S_(T2D1)), (S_(T1D2)) and (S_(T2D2)) by using measuredsignals (S_(D1)) and (S_(D2)).

Step (c): Establishing Additional Equations

Afterwards, provided are two additional equations each of whichcomprises at least one variable selected from the group consisting ofthe four variables, (S_(T1D1)), (S_(T2D1)), (S_(T1D2)) and (S_(T1D2)).

Following setting up the two equations (I) and (II), two additionalequations are established such that a total of the four equations areprovided for obtaining solutions to at least one of the variables(particularly at least two of the variables, more particularly all ofthe variables) in the equations (I) and (II) by a mathematicalprocessing.

For instance, where the two additional equations enable that theequations (I) and (II) have the same variable set (i.e., the equations(I) and (II) are converted to a simultaneous equation), the solution toat least one of the variables in the equations (I) and (II) can beobtained by solving the simultaneous equation (particularly, linearsimultaneous equation with two variables). Alternatively, the solutionto at least one of the variables in the equations (I) and (II) may beobtained in a different manner from the simultaneous equation-solvingapproach.

According to an embodiment, the two additional equations are selectedfrom the group consisting of the following equations:f(S _(T1D1) ,S _(T1D2))=RV_(T1(D1D2))  (III),f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2))  (IV), andS _(T1D2)=an arbitrarily selected value  (V)

wherein, RV_(t1(D1D2)) is a reference value (RV) of the first targetnucleic acid sequence (T1) representing a relationship of change insignals provided by the first signal-generating means at the firstdetection temperature and the second detection temperature,RV_(T2(D1D2)) is a reference value (RV) of the second target nucleicacid sequence (T2) representing a relationship of change in signalsprovided by the second signal-generating means at the first detectiontemperature and the second detection temperature; f(S_(T1D1), S_(T1D2))represents a function of S_(T1D1) and S_(T1D2); f(S_(T2D1), S_(T2D2))represents a function of S_(T2D1) and S_(T2D2);

wherein S_(T1D2)=an arbitrarily selected value in the equation (V) maybe selected with a proviso that the first signal-generating means isprepared to generate no signal in the presence of the first targetnucleic add sequence at the second detection temperature.

According to an embodiment of the present invention, when the equation(V) is selected, the equation (III) is not selected as an additionalequation.

The above-described embodiment describes two approaches to obtain thesolution to at least one of the variables in the equations (I) and (II).

The first approach is to use the equations (III) and (IV) as the twoadditional equations.

Interestingly, the present inventors have found that when signalsindicating the presence of a target nucleic add sequence are detected ina single reaction vessel at predetermined two detection temperatures,there is a signal change in a certain relationship (pattern or rule).For example, a signal change between a signal detected at the relativelyhigh detection temperature and a signal detected at the relatively lowdetection temperature for a target nucleic add sequence shows a certainrelationship (pattern or rule). For example, the intensities of thesignals may be identical or substantially identical to each other or theintensities of the signals may be different from each other but in acertain range at the two detection temperatures.

The present invention is to adopt such findings to obtaining referencevalues and differentiating signals of interest for target nucleic acidsequences. Because signals for a target nucleic acid sequence aredetected with differing only detection temperatures (e.g. no change ofamount of the target or no variation of buffer conditions), there is acertain relationship (pattern or rule) in a signal change between thetwo detection temperatures. According to an embodiment, the presentmethod is performed under conditions that permit a certain relationship(pattern or rule) in a signal change for a target nucleic acid sequencebetween the two detection temperatures.

“Reference value (RV)” of a target nucleic add sequence represents arelationship of change in signals provided by a signal-generating meansfor detection of the target nucleic acid sequence means at two detectiontemperature.

According to an embodiment, where the RV value is used, a signal at oneof detection temperatures may be expressed in terms of a signal at theother detection temperature.

In the equations (III) and (IV), RV_(T1(D1D2)) and RV_(T2(D1D2)) as areference value are a predetermined value. According to an embodiment,the reference value may be obtained by using a standard materialcorresponding to a target nucleic acid sequence. For instance, thestandard material corresponding to a target nucleic acid sequence isincubated with a corresponding signal-generating means and then signalsare detected at the first detection temperature and the second detectiontemperature, followed by obtaining a relationship of change in thesignals at the first detection temperature and the second detectiontemperature. The relationship of change in signals may be expressed asdifference between signals detected at the first detection temperatureand the second detection temperature.

The reference value is obtained through a mathematical processing ofsignals provided by the signal-generating means at the first detectiontemperature and the second detection temperature. Such mathematicalprocessing is a function of the signals. The function used in obtainingreference values include any function so long as it gives a relationshipof change in signals provided by the signal-generating means at thefirst detection temperature and the second detection temperature. Forinstance, the function may be presented as a mathematical processingsuch as addition, multiplication, subtraction and division of signals.

The characteristics of the signals provided at the first detectiontemperature and the second detection temperature per se may be used toobtain a relationship of change in signals at the first detectiontemperature and the second detection temperature. Alternatively, thesignals at the first detection temperature and the second detectiontemperature may be modified by mathematically processing thecharacteristics of the signals and used to obtain the relationship ofchange in signals at the first detection temperature and the seconddetection temperature.

Alternatively, the initially obtained reference value may be modifiedand used a reference value.

According to an embodiment, the term “signal” with conjunction with thereference value includes not only signals per se obtained at detectiontemperatures but also a modified signal provided by mathematicallyprocessing the signals.

As described above, because RV_(T1(D1D2)) and RV_(T2(D1D2)) as areference value are a predetermined value, RV_(T1(D1D2)) andRV_(T2(D1D2)) may serve as a convertor for (i) converting the twovariables (S_(T1D1)) and (S_(T1D2)) in the equations (I) and (II) into avariable selected from the two variables and (ii) converting the twovariables (S_(T2D1)) and (S_(T2D2)) in the equations (I) and (II) into avariable selected from the two variables.

According to an embodiment, the equation (III) is used to convert thetwo variables (S_(T1D1)) and (S_(T1D2)) in the equations (I) and (U)into a variable selected from the two variables, and the equation (IV)is used to convert the two variables (S_(T2D1)) and (S_(T2D2)) in theequations (I) and (II) into a variable selected from the two variables.

According to an embodiment, one of the two additional equations isselected from the equation (III) and the other is selected from theequation (IV).

The reference value used in this invention may be obtained in variousmanners. For instance, the reference value may be given as ananticipated value. In considering a target sequence, a signal-generatingmeans and detection temperatures, the reference value representing arelationship of change in signals at the first detection temperature andthe second detection temperature may be anticipated. Alternatively, thereference value may be given as an experimental or practical value byperforming experiments for obtaining the reference value.

According to an embodiment, (i) RV_(T1(D1D2)) is obtained by (i-1)incubating the first target nucleic acid sequence with the firstsignal-generating means for detection of the first target nucleic addsequence, (i-2) detecting signals at the first detection temperature andthe second detection temperature, and (i-3) then obtaining a differencebetween the signals detected at the first detection temperature and thesecond detection temperature, and (ii) RV_(T2(D1D2)) is obtained by(ii-1) incubating the second target nucleic add sequence with the secondsignal-generating means for detection of the second target nucleic addsequence, (ii-2) detecting signals at the first detection temperatureand the second detection temperature, and (ii-3) then obtaining adifference between the signals detected at the first detectiontemperature and the second detection temperature; wherein RV_(T1(D1D2))is different from RV_(T2(D1D2)).

The term “difference between signals detected at the first detectiontemperature and the second detection temperature” in obtaining areference value is an embodiment of a relationship of change in signalsat the first detection temperature and the second detection temperature.

According to an embodiment, the difference between the signals detectedat the first detection temperature and the second detection temperaturecomprises a difference to be obtained by mathematically processing thesignal detected at the first detection temperature and the signaldetected at the second detection temperature.

According to an embodiment, where the mathematical processing is done,the characteristics of the signal should be vulnerable to themathematical processing. In certain embodiment, the mathematicalprocessing includes calculation (e.g., addition, multiplication,subtraction and division) using signals or obtaining other valuesderived from signals.

The difference between the signals at the first detection temperatureand the second detection temperature may be expressed in variousaspects. For example, the difference may be expressed as numericalvalues, the presence/absence of signal or plot with signalcharacteristics.

The mathematical processing of the signals for obtaining the differencemay be carried out by various calculation methods and theirmodifications.

In particular, the mathematical processing of the signals for obtainingthe difference may be carried out by calculating a ratio between signalsat the first detection temperature and the second detection temperature.

For instance, the ratio of the end-point intensity of the signaldetected at the second detection temperature to the end-point intensityof the signal detected at the first detection temperature may be used asreference values.

According to an embodiment of this invention, the mathematicalprocessing of the signals to obtain the difference between the signalsis a calculation of a ratio of the signal detected at the relatively lowdetection temperature to the signal detected at the relatively highdetection temperature. According to an embodiment of this invention, themathematical processing of the signals to obtain the difference betweenthe signals is a calculation of a ratio of the signal detected at therelatively high detection temperature to the signal detected at therelatively low detection temperature.

According to an embodiment, a reference value may be obtained bycalculating the subtraction between the signal detected at therelatively high detection temperature and the signal detected at therelatively low detection temperature.

The mathematical processing for obtaining the difference may be carriedout in various fashions. The mathematical processing may be carried outby use of a machine. For example, the signals may be undergone amathematical processing by a processor in a detector or real-time PCRdevice. Alternatively, the signals may be manually undergone amathematical processing particularly according to a predeterminedalgorithm.

According to an embodiment of the present invention, RV_(T1(D1D2)) isdifferent from RV_(T2(D1D2)). According to an embodiment of the presentinvention, the first signal-generating means and the secondsignal-generating means are designed such that RV_(T1(D1D2)) isdifferent from RV_(T2(D1D2)).

Where the RV values are different from each other, a quantitativeexpression describing a difference extent may be varied depending onapproaches for calculating the RV values.

According to an embodiment, signals for obtaining reference values maybe processed by a common calculation method to provide a reference valuefor comparison, and then a difference extent between two referencevalues may be obtained by using the reference value for comparison.According to an embodiment, the common calculation method is division oftwo signals.

For instance, while two signals are processed by subtraction forobtaining reference values used to analyze signals according to thepresent method, the two signals may be processed by division forobtaining a reference value for comparison.

According to an embodiment, RV_(T1(D1D2)) is at least 1.1-fold,1.2-fold, 1.3-fold, 1.5-fold, 1.7-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, 4-fold, 5-fold or 10-fold larger than RV_(T2(D1D2)).Alternatively, RV_(T2(D1D2)) is at least 1.1-fold, 1.2-fold, 1.3-fold,1.5-fold, 1.7-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-foldor 10-fold larger than RV_(T1(D1D2)).

According to an embodiment, where the comparison is performed todetermine whether the first reference value is different from the secondreference, the reference values are calculated by division of thesignals. According to an embodiment, the method of calculating thereference value for determining whether the first reference value isdifferent from the second reference may be the same or different fromthe method of calculating the reference value for detecting the targetnucleic acid sequence.

According to an embodiment of this invention, the signal-generatingmeans for the reference value may be the same as that for the detectionof the target nucleic acid sequence.

According to an embodiment, the incubation conditions for obtaining thereference values are the same as those for analysis of the sample.

For a target nucleic acid sequence, the reference values may be obtainedin various reaction conditions including the amounts of components (e.g.the target nucleic add sequence, signal-generating means, enzymes anddNTPs), buffer pH or reaction time. According to an embodiment of thisinvention, the reference value may be obtained under reaction conditionssufficient to provide a saturated signal at the reaction completion.According to an embodiment of this invention, the difference between thesignals obtained in obtaining the reference value has a certain rangeand the reference value is selected within the certain range or withreferring to the certain range. According to an embodiment of thisinvention, the reference value may be selected with maximum or minimumvalue of the certain range or with referring to maximum or minimum valueof the certain range. Particularly, the reference value may be modifiedin considering standard variation of the reference values obtained invarious conditions, acceptable error ranges, specificity or sensitivity.

f(S_(T1D1), S_(T1D2)) or f(S_(T2D1), S_(T2D2)) in “f(S_(T1D1),S_(T1D2))=RV_(T1(D1D2)) or f(S_(T2D1), S_(T2D2))=RV_(T2(D1D2))” may bepresented by a mathematical expression for calculating RV_(T1(D1D2)) orRV_(T2(D1D2)).

According to an embodiment, f(S_(T1D1), S_(T1D2))=RV_(T1(D1D2))comprises S_(T1D1)/S_(T1D2)=RV_(T1D1/D2) (VI) orS_(T1D2)/S_(T1D1)=RV_(T1D2/D1) (VII); and f(S_(T2D1),S_(T2D2))=RV_(T2(D1D2)) comprises S_(T2D1)/S_(T2D2)=RV_(T2D1/D2) (VIII)or S_(T2D2)/S_(T2D1)=RV_(T2D2/D1) (IX).

Where RV is obtained by using a ratio between signals at the firstdetection temperature (D1) and the second detection temperature (D2),f(S_(T1D1), S_(T1D2)) or f (S_(T2D1), S_(T2D2)) may be presented by amathematical expression for calculating ratio between signals. In theterm “RV_(T1(D1D2))”, T1(D1D2) indicates that a calculated RVcorresponds to the reference value for the first target nucleic acidsequence (T1) obtained by calculating a difference between signals atthe first detection temperature (D1) and the second detectiontemperature (D2). Upon determining a method for calculation RV, the term“T1(D1D2)” may be specifically expressed in considering the calculationmethod.

For example, the term “RV_(T1D1/D2)” means a reference value for thefirst target nucleic acid sequence (T1) obtained by calculating a ratioof a signal detected at the first detection temperature (D1) to a signaldetected at the second detection temperature (D2). Alternatively, theterm “RV_(T1D2/D1)” means a reference value for the first target nucleicacid sequence (T1) obtained by calculating a ratio of a signal detectedat the second detection temperature (D2) to a signal detected at thefirst detection temperature (D1).

According to an embodiment, RV_(T1D1/D2) is obtained by (i-1) incubatingthe first target nucleic acid sequence with the first signal-generatingmeans for detection of the first target nucleic acid sequence, (i-2)detecting signals at the first detection temperature and the seconddetection temperature, and (i-3) then calculating a ratio of a signaldetected at the first detection temperature to a signal detected at thesecond detection temperature; RV_(T1D2/D1) is obtained by performing thesteps (i-1) and (i-2) and then calculating a ratio of the signaldetected at the second detection temperature to the signal detected atthe first detection temperature; RV_(T2D1/D2) is obtained by (ii-1)incubating the second target nucleic acid sequence with the secondsignal-generating means for detection of the second target nucleic acidsequence, (ii-2) detecting signals at the first detection temperatureand the second detection temperature, and then (ii-3) calculating aratio of the signal detected at the first detection temperature to thesignal detected at the second detection temperature; and RV_(T2D2/D1) isobtained by performing the steps (ii-1) and (ii-2) and then calculatinga ratio of the signal detected at the second detection temperature tothe signal detected at the first detection temperature.

According to an embodiment, the two additional equations for obtaining(S_(T1D1)), (S_(T1D2)), (S_(T2D1)) and (S_(T2D2)) comprises one of thefollowing equations (VI) and (VII) and one of the following equations(VIII) and (IX):S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)  (VI)S _(T1D2) /S _(T1D1)=RV_(T1D2/D1)  (VII)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)  (VIII)S _(T2D2) /S _(T2D1)=RV_(T2D2/D1)  (IX).

According to an embodiment, a plurality of equations for calculating RVfor a certain target nucleic acid sequence at selected two detectiontemperatures may be presented (e.g. the equation (VI) and (VII) for thefirst target nucleic acid sequence (T1) at the first detectiontemperature (D1) and the second detection temperature (D2)). Accordingto an embodiment, in such case, only one equation among a plurality ofequations is selected as one of the additional equations.

The equations (III) and (IV) together with the equations (I) and (II)are used for obtaining solutions to at least one of the variables. Asexemplified in Example 1 and FIGS. 1A-1D, where f(S_(T1D1), S_(T1D2)) isS_(T1D1)/S_(T1D2) and f(S_(T2D1), S_(T2D2)) is S_(T2D1)/S_(T2D2), theequation set for obtaining solutions to variables includes thefollowing:S _(T1D1) +S _(T2D1) =S _(D1)S _(T1D2) +S _(T2D2) =S _(D2)S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)Because RV_(T1D1/D2) and RV_(T2D1/D2) are an experimentallypredetermined value, the solutions to the variables, (S_(T1D1)),(S_(T2D1)), (S_(T1D2)) and (S_(T1D2)) may be obtained by the fourequations.

Alternatively, a reference value may be obtained by calculating thesubtraction between the signal detected at the first detectiontemperature and the signal detected at the second detection temperature.

The second approach to obtain the solution to at least one of thevariables in the equations (I) and (II) is to use the equations [(III)and (V)] or [(IV) and (V)] as the two additional equations. S_(T1D2)=anarbitrarily selected value in the equation (V) may be selected with aproviso that the first signal-generating means is prepared to generatesubstantially no signal in the presence of the first target nucleic acidsequence at the second detection temperature.

Where the equation (V) is applied, an arbitrarily selected value may beused as S_(T1D2). According to an embodiment, it is convenient in termsof performance of the present invention that zero (0) may be used asS_(T1D2) (S_(T1D2)=0). Alternatively, either negative numeric constantor positive numeric constant may be used as S_(T1D2) (S_(T1D2)=negativenumeric constant or S_(T1D2)=positive numeric constant).

The numeric values of solutions to the variables except for (S_(T1D2))may be varied depending on values of the constants used in the equation(V). However, it is noteworthy that the numeric values of the solutionsto the variables have values calculated by using numeric values of theconstants selected as S_(T1D2). Alternatively, such approach for solvingthe variables may be expressed in that the numeric values of thesolutions to the variables except for (S_(T1D2)) are results obtained byusing the constants selected as a value of S_(T1D2) used for signalextraction.

Therefore, a signal differentiation for each target nucleic acidsequence is enabled at each detection temperature even when any constantis applied in the equation (V), while calculated variables becomedifferent depending on the constants.

In particular, when signals are detected at a plurality of cycles forobtaining a data set, a signal differentiation at each cycle isperformed and then the results of signal differentiation are plottedover all cycles, absolute signal values may be altered depending on theconstants in the equation (V). However, occurrence or non-occurrence ofsignal change (e.g., signal change pattern) over all cycles remainsunchanged such that the signal differentiation for target detection maybe successfully accomplished.

According to an embodiment, the solutions to the variables calculated byusing the equation (V) may be mathematically modified in a definedmanner. In such modification, the solutions to the variables may bemodified to have values calculated by using other constants (e.g.,S_(T1D2)=0).

The numeric values of the constants in the equation (V) used forobtaining solutions to variables are not considerable when the presenceor absence of a target nucleic acid sequence of interest is determined.

When the presence or absence of a target nucleic acid sequence ofinterest is determined by using solutions to variables calculated byusing the equation (V) as an additional equation, (i) the solutions tothe variables calculated per se or (ii) the solutions mathematicallymodified in a defined manner may be used. Additionally, a threshold fordetermining significance of the calculated solutions or theirmathematical modifications may be established in considering the numericvalues of the constants in equation (V), thereby finally determining thepresence or absence of a target nucleic acid sequence of interest.

According to an embodiment, when the equation (V) is selected, theequation (III) is not selected but the equation (IV) is selected.

It would be understood by one of skill in the art that the descriptionsfor the second approach in the Specification and Claims encompassembodiments in which S_(T1D1)=an arbitrarily selected value, S_(T2D1)=anarbitrarily selected value, or S_(T2D2)=an arbitrarily selected value isselected. For example, S_(T2D2)=an arbitrarily selected value may beselected with a proviso that the second signal-generating means isprepared to generate substantially no signal in the presence of thesecond target nucleic acid sequence at the second detection temperature.When the equation S_(T2D2)=an arbitrarily selected value is selected,the equation (IV) is not selected but the equation (III) is selected.Such various embodiments may be claimed and covered by the descriptionof an embodiment (S_(T1D2)=an arbitrarily selected value) with avoidingundue complexity of the Specification by unnecessary redundancy.

The term “generate no signal” includes not only “no generation ofsignal” but also “signal generation with no significance” such as abackground signal.

In particular, when signals are detected at a plurality of cycles forobtaining a data set, the term “generate no signal” means that there isno signal change or no significant signal change between cycles.

The case in which signals are detected at a plurality of cycles forobtaining a 5 data set may include, but not limited to, obtaining asignal amplification curve by amplification reaction such as real-timePCR.

According to an embodiment, the two additional equations for obtaining(S_(T1D1)), (S_(T1D2)), (S_(T2D1)) and (S_(T2D2)) comprises one of thefollowing equations (VIII) and (IX) and the following equation (V):S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)  (VIII)S _(T2D2) /S _(T2D1)=RV_(T2D2/D1)  (IX)S _(T1D2)=an arbitrarily selected value  (V).

As exemplified in Example 2 and FIGS. 2C-2D, where the firstsignal-generating means is prepared to generate substantially no signalin the presence of the first target nucleic acid sequence at the seconddetection temperature, S_(T1D2)=0 in the equation (V) and f(S_(T2D1),S_(T2D2))=RV_(T2(D1D2)) (IV) as the additional equation may be selectedfor obtaining solutions to at least one of the variables. Wheref(S_(T2D1), S_(T2D2)) is S_(T2D1)/S_(T2D2), the equation set forobtaining solutions to variables includes the following:S _(T1D1) +S _(T2D1) =S _(D1)S _(T1D2) +S _(T2D2) ⁼ S _(D2)S _(T1D2)=0S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)

Because RV_(T2D1/D2) is a predetermined constant value, the solutions tothe variables, (S_(T1D1)), (S_(T2D1)) and (S_(T1D2)) may be obtained bythe four equations.

As a particular embodiment of the first approach using the equations(III) and (IV) as the two additional equations, RV_(T1(D1D2)) may beselected as an arbitrarily selected value when the firstsignal-generating means is prepared to generate no signal in thepresence of the first target nucleic acid sequence at the seconddetection temperature.

According to an embodiment, when the first signal-generating means isprepared to generate substantially no signal in the presence of thefirst target nucleic acid sequence at the second detection temperature,RV_(T1(D1D2)) is an arbitrarily selected value.

As described above, where the first target nucleic acid sequence (T1)generates no signal at the second detection temperature (D2), anarbitrarily selected value may be used as S_(T1D2), wherebyRV_(T1(D1D2)) may become an arbitrarily selected value.

Particularly, the arbitrarily selected value as RV_(T1(D1D2)) may beselected such that a solution (value) of S_(T1D2) is rendered to be avalue or around the value (e.g., “0” or around “0” in the equations.

The term “around the value” with conjunction to “a value or around thevalue” means any number within a significance level being considerableas a value.

Particularly, when f(S_(T1D1), S_(T1D2)) is be presented by amathematical expression for calculating ratio between signals at thefirst detection temperature (D1) and the second detection temperature(D2), RV_(T1(D1D2)) may be selected as an arbitrarily selected value.

Practically, even when the first signal-generating means is prepared togenerate no signal in the presence of the first target nucleic acidsequence at the second detection temperature, it is usually to generatesignals with very weak intensities (e.g., background signal). Inparticular, the arbitrarily selected value as RV_(T1(D1D2)) may be thesame as, less or greater than RV_(T1(D1D2)) calculated withpractically-obtained signal values.

It would be understood by one of skill in the art that the descriptionsfor the first approach in the Specification and Claims encompassembodiments in which RV_(T1(D1D2)) or RV_(T2(D1D2)) is an arbitrarilyselected value. For example, RV_(T2(D1D2)) may be an arbitrarilyselected value with a proviso that the second signal-generating means isprepared to generate substantially no signal in the presence of thesecond target nucleic acid sequence at the second detection temperature.Such various embodiments may be claimed and covered by the descriptionof the embodiment (RV_(T1(D1D2)) as an arbitrarily selected value) withavoiding undue complexity of the Specification by unnecessaryredundancy.

The equations (III) and (IV) together with the equations (I) and (II)are used for obtaining solutions to at least one of the variables. Asexemplified in Example 2 and FIGS. 2G-2H, where the firstsignal-generating means is prepared to generate substantially no signalin the presence of the first target nucleic acid sequence at the seconddetection temperature, RV_(T1(D1D2)) may be an arbitrarily selectedvalue. Where f (S_(T1D1), S_(T1D2)) is S_(T1D1)/S_(T1D2) and f(S_(T2D1),S_(T2D2)) is S_(T2D1)/S_(T2D2), the equation set for obtaining solutionsto variables includes the following:S _(T1D1) +S _(T2D1) =S _(D1)S _(T1D2) +S _(T2D2) =S _(D2)S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)

Because RV_(T1D1/D2) is an arbitrarily selected value, the solutions tothe variables, (S_(T1D1)), (S_(T2D1)), (S_(T1D2)) and (S_(T1D2)) may beobtained by the four equations.

It is very interesting that the present method allows for quantificationof a signal amount (e.g. signal intensity) generated by each of thesignal-generating means. As described above, the solutions to thevariables, (S_(T1D1)), (S_(T2D1)), (S_(T1D2)) and (S_(T1D2)) permitquantification of a signal amount (e.g. signal intensity) generated byeach of the signal-generating means, which may be applied toquantification of the target nucleic acid sequences.

Step (d): Obtaining Solutions to Variables

Finally, the solutions to at least one of the variables are obtained byusing the four equations provided in the steps (b) and (c) fordifferentiating at least one of the signals of interest to be assignedto at least one of the two target nucleic acid sequences.

As described above, the four equations provided in the steps (b) and (c)are used to obtain the solutions to at least one of the variables fordifferentiating at least one of the signals of interest to be assignedto at least one of the two target nucleic acid sequences, whereby thepresence of at least one of the two target nucleic acid sequences may bedetermined.

Particularly, the solutions to at least two (more particularly at leastthree, most particularly four) of the variables are obtained by usingthe four equations provided in the steps (b) and (c) for differentiatingat least two (more particularly at least three, most particularly four)of the signals of interest to be assigned to at least one (mostparticularly all) of the two target nucleic acid sequences.

According to an embodiment, the present method is used to obtainsolutions (values) to the two variables and one of the two variables isselected from the variables for the first target nucleic acid sequenceand the other is selected from the variables for the second targetnucleic acid sequence.

According to an embodiment, depending on approaches for generatingsignals, a threshold value may be employed to analyze whether theobtained solutions (values) to the variables may be significant.

A negative control, sensitivity or label used may be considered fordetermining the threshold value. According to an embodiment of thisinvention, a threshold value may be determined by user or automatically.

According to an embodiment, the threshold value is determined withreferring to signals detected using only the first target nucleic acidsequence or only the second target nucleic acid sequence.

According to an embodiment, where signals are generated in a real-timemanner associated with target amplification by PCR, the signals at eachamplification cycle or some selected cycles are mathematically processedwith the reference values and the calculation results are plottedagainst cycles and used for differentiating the signals of interest(e.g., determination of the presence of the target nucleic acidsequence).

According to an embodiment of this invention, the two target nucleicacid sequences comprises a nucleotide variation and one of the twotarget nucleic acid sequences comprises one type of the nucleotidevariation and the other comprises the other type of the nucleotidevariation.

The term “nucleotide variation” used herein refers to any single ormultiple nucleotide substitutions, deletions or insertions in a DNAsequence at a particular location among contiguous DNA segments that areotherwise similar in sequence. Such contiguous DNA segments include agene or any other portion of a chromosome. These nucleotide variationsmay be mutant or polymorphic allele variations. For example, thenucleotide variation detected in the present invention includes SNP(single nucleotide polymorphism), mutation, deletion, insertion,substitution and translocation. Exemplified nucleotide variationincludes numerous variations in a human genome (e.g., variations in theMTHFR (methylenetetrahydrofolate reductase) gene), variations involvedin drug resistance of pathogens and tumorigenesis-causing variations.The term nucleotide variation used herein includes any variation at aparticular location in a nucleic acid sequence. In other words, the termnucleotide variation includes a wild type and its any mutant type at aparticular location in a nucleic acid sequence.

According to an embodiment of this invention, the nucleotide variationdetected by the present invention is a SNP (single nucleotidepolymorphism).

According to an embodiment of this invention, a homozygote composed of afirst SNP allele is detected by using the signal-generating means forthe first target nucleic acid sequence, a homozygote composed of asecond SNP allele by using the second signal-generating means for thesecond target nucleic acid sequence and a heterozygote composed of thefirst SNP allele and the second SNP allele by using the twosignal-generating means.

II. Differentiation of Signals of Interest for at Least Three TargetNucleic Acid Sequences

In another aspect of this invention, there is provided a method fordifferentiating signals of interest for each of target nucleic acidsequences in the number of N in a sample, which are not differentiableby a single type of detector, comprising:

(a) incubating the sample with signal-generating means in the number ofN for detection of the target nucleic acid sequences in the number of Nand detecting signals at detection temperatures in the number of N;wherein each of the target nucleic acid sequences is detected by acorresponding signal-generating means; wherein the signals of interestto be generated by the signal-generating means in the number of N arenot differentiated for each target nucleic acid sequence by a singletype of detector; wherein N is an integer not less than 2;

(b) providing the following equations in the number of N each of whichcomprises variables representing the signals of interest generated ateach detection temperature for the target nucleic acid sequences;

$\begin{matrix}{{S_{T\; 1D\; 1} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 1}}}} = S_{D\; 1}} & (1) \\{{S_{T\; 1D\; 2} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 2}}}} = {S_{D\; 2}\mspace{76mu} \cdot \mspace{275mu} \cdot \mspace{76mu} \cdot \mspace{275mu} \cdot \mspace{76mu} \cdot \mspace{275mu} \cdot}} & (2) \\{{S_{T\; 1{DN}} + S_{T\; 2{DN}} + {\cdot \cdot \cdot \cdot \cdot {+ S_{TNDN}}}} = S_{DN}} & (N)\end{matrix}$

wherein each of (S_(D1)) to (S_(DN)) is a signal detected at eachdetection temperature; each of (S_(T1D1)) to (S_(TNDN)) is a variablerepresenting a signal of interest generated by each signal-generatingmeans at each detection temperature; and the total number of thevariables is N²;

(c) providing additional equations in the number of (N²−N) each of whichcomprises at least one variable selected from the group consisting ofthe variables, (S_(T1D1)) to (S_(TNDN)); and

(d) obtaining solutions to at least one of the variables by theequations in the number of N² provided in the steps (b) and (c) fordifferentiating at least one of the signals of interest to be assignedto at least one of the target nucleic acid sequences in the number of N.

The present method is expressed herein as a method for differentiatingsignals of interest for each of target nucleic acid sequences in thenumber of N in a sample and alternatively may be expressed as a methodfor detecting at least one of the target nucleic acid sequences in asample or a method for determining the presence of at least one of thetarget nucleic acid sequences in a sample by assigning at least one ofsignals of interest to at least one of the target nucleic acidsequences.

Since the second aspect follows in principle the technological principleand features of the first aspect of this invention described above, thecommon descriptions between them are omitted in order to avoid undueredundancy leading to the complexity of this specification. Whenreferring to descriptions for the first aspect in order to describe thesecond aspect, it should be noted that the second aspect may be, inpart, different from the first aspect. Therefore, it would be understoodto those skilled in the art that some descriptions for the first aspectmay be directly applied to descriptions for the second aspect and otherdescriptions may be applied to descriptions for the second aspect by alittle modification.

The present invention will be described in more detail as follows:

Step (a): Incubating Samples with Signal-Generating Means and SignalDetection

Firstly, the sample to be analyzed is incubated with signal-generatingmeans in the number of N for detection of the target nucleic acidsequences in the number of N and then signals from the signal-generatingmeans are detected at detection temperatures in the number of N; whereineach of the target nucleic acid sequences is detected by a correspondingsignal-generating means; wherein the signals of interest to be generatedby the signal-generating means in the number of N are not differentiatedfor each target nucleic acid sequence by a single type of detector;wherein N is an integer not less than 2.

The number of the target nucleic acid sequences (N) to be detected inthe present invention is not limited, including more than 2, 3, 4, 5, 6,7, 8, 9 and 10 target nucleic acid sequences.

For example, where the number of the target nucleic acid sequences isthree, N is three and the target nucleic acid sequence may be expressedas T1, T2 and T3, respectively.

According to an embodiment, where the target nucleic acid sequences arein the number of N, the signal-generating means are used in the numberof N.

Where the target nucleic acid sequences are in the number of N, thedetection temperatures are used in the number of N. For example, wherethe number of the target nucleic acid sequences is three, N is three andthe detection temperatures are used in the number of three. Thedetection temperatures may be expressed as D1, D2 and D3, respectively.

The present invention utilizes signal-generating means for providingsignals for target nucleic acid sequences. Each of the target nucleicacid sequences is detected by a corresponding signal-generating means.For example, the g^(th) target nucleic acid sequence among the targetnucleic acid sequences in the number of N may be detected by the g^(th)signal-generating means, where the term “g” is an integer selected from1 to N.

According to an embodiment of this invention, the incubation ispreformed under conditions allowing a signal generation by thesignal-generation means.

According to an embodiment of this invention, the incubation ispreformed in a single reaction vessel containing signal-generating meansin the number of N.

The signal-generation means for the second aspect of the presentinvention may be with reference to those for the first aspect of thepresent invention.

In the present invention, signals may be generated by using variousmaterials in various fashions.

According to an embodiment, at least one of the signal-generating meansin the number of N is a signal-generating means to generate a signal ina dependent manner on the formation of a duplex.

According to an embodiment, the signal-generating means for each of thetarget nucleic acid sequences are signal-generating means to generate asignal in a dependent manner on the formation of a duplex.

Particularly, the signal is generated by the formation of a duplexbetween a target nucleic acid sequence and a detection oligonucleotidespecifically hybridized with the target nucleic acid sequence.

Particularly, the signal is generated by a duplex formed in a dependentmanner on cleavage of a mediation oligonucleotide specificallyhybridized with the target nucleic acid sequence.

The signal by the duplex formed in a dependent manner on cleavage of themediation oligonucleotide may be generated by various methods, includingPTOCE (PTO cleavage and extension) method (WO 2012/096523), PCE-SH (PTOCleavage and Extension-Dependent Signaling OligonucleotideHybridization) method (WO 2013/115442) and PCE-NH (PTO Cleavage andExtension-Dependent Non-Hybridization) method (PCT/KR2013/012312).

According to an embodiment, at least one of the signal-generating meansin the number of N is a signal-generating means to generate a signal ina dependent manner on cleavage of a detection oligonucleotide.

According to an embodiment, the signal-generating means for each of thetarget nucleic acid sequences are signal-generating means to generate asignal in a dependent manner on cleavage of a detection oligonucleotide.

Particularly, the signal is generated by hybridization of the detectionoligonucleotide with a target nucleic acid sequence and then cleavage ofthe detection oligonucleotide. The signal by hybridization of thedetection oligonucleotide with a target nucleic acid sequence and thencleavage of the detection oligonucleotide may be generated by variousmethods, including TaqMan probe method (U.S. Pat. Nos. 5,210,015 and5,538,848).

Particularly, the signal is generated by cleavage of the detectionoligonucleotide in a dependent manner on cleavage of a mediationoligonucleotide specifically hybridized with the target nucleic acidsequence. According to an embodiment of this invention, the cleavage ofa mediation oligonucleotide releases a fragment and the fragmentmediates a formation of a duplex or a cleavage of a detectionoligonucleotide by an extension of the fragment on a captureoligonucleotide.

According to an embodiment of this invention, the signal-generatingmeans for at least one of the target nucleic acid sequences in thenumber of N is a signal-generating means by cleavage of a detectionoligonucleotide, and the signal-generating means for the other targetnucleic acid sequences are a signal-generating means by the formation ofa duplex.

According to an embodiment, each target nucleic acid sequence isdetected by a signal-generating means capable of generating signals atall detection temperatures.

According to an embodiment, an g^(th) target nucleic acid sequence amongthe target nucleic acid sequences in the number of N is detected by asignal-generating means capable of generating signals at detectiontemperatures in the number of at least “g”. For example, an 3^(rd)target nucleic acid sequence among the target nucleic acid sequences isdetected by a signal-generating means capable of generating signals atthree or more detection temperatures.

According to an embodiment of this invention, the signal-generatingmeans in the number of N comprise an identical label and signals fromthe label are not differentiated by the single type of detector.

According to an embodiment of this invention, the step (a) is performedin a signal amplification process concomitantly with a nucleic acidamplification.

In the present invention, the signal generated by signal-generatingmeans may be amplified simultaneously with target amplification.Alternatively, the signal may be amplified with no target amplification.

According to an embodiment of this invention, the signal generation isperformed in a process involving signal amplification together withtarget amplification.

According to an embodiment of this invention, the target amplificationis performed in accordance with PCR (polymerase chain reaction).

According to an embodiment of this invention, the incubation ispreformed in the conditions allowing target amplification well as signalgeneration by the signal-generation means.

According to an embodiment of this invention, the step (a) is performedin a signal amplification process without a nucleic acid amplification.

Where the signal is generated by methods including cleavage of anoligonucleotide, the signal may be amplified with no targetamplification.

During or after the incubation (reaction) of the sample with thesignal-generating means to provide signals, the signals are detected byusing a detector such as a single type of detector. According to thepresent invention, the presence or absence of the two target nucleicacid sequences may be determined even using a single type of detector.

The present invention may be performed by using any type ofsignal-generating means in view of detection temperatures.

The signal-generating means may be designed to provide signals at all ora portion of the detection temperatures in the number of N.

Particularly, the signal-generating means may be designed to providedifferent signals upon changing detection temperatures.

According to an embodiment, the detection temperatures are predeterminedin considering a temperature range to allow signal generation by thesignal-generating means.

A detection temperature is selected from the temperature range to allowsignal generation by the signal generation mean.

According to an embodiment, the detection temperatures in the number ofN are different from each other.

According to an embodiment, the signal-generating means for detecting atarget nucleic acid sequence may be signal-generating means to providedifferent signals (e.g. signal intensity) from each other at each of thedetection temperatures in the number of N.

According to an embodiment, signal-generating means for the two targetnucleic acid sequences are firstly constructed and then detectiontemperatures for the two target nucleic acid sequences are allocated,followed by performing the step (a).

According to an embodiment of this invention, when the signal-generatingmeans generates a signal in a dependent manner on cleavage of adetection oligonucleotide, the detection temperature is arbitrarilyselected. In other words, any temperature can be selected so long as thesignal generated by cleavage of a detection oligonucleotide may bedetected. As described above, where the signal is generated beingdependent manner on cleavage of the detection oligonucleotide, the labelreleased by the cleavage may be detected at various temperatures.

The detector used in the present invention includes any means capable ofdetecting signals. For example, where fluorescent signals are used,photodiodes suitable in detection of the fluorescent signals may beemployed as detectors. The detection using a single type of detectormeans that the detection is performed by using a detector capable ofdetecting a single type of signal or using each channel (i.e.,photodiode) of a detector carrying several channels (i.e., photodiodes).

Step (b): Establishing Equations Comprising Variables RepresentingSignals of Interest

Provided are the following equations in the number of N each of whichcomprises variables representing the signals of interest generated ateach detection temperature for the target nucleic acid sequences;

$\begin{matrix}{{S_{T\; 1D\; 1} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 1}}}} = S_{D\; 1}} & (1) \\{{S_{T\; 1D\; 2} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 2}}}} = {S_{D\; 2}\mspace{211mu} \cdot \mspace{211mu} \cdot \mspace{211mu} \cdot}} & (2) \\{{S_{T\; 1{DN}} + S_{T\; 2{DN}} + {\cdot \cdot \cdot \cdot \cdot {+ S_{TNDN}}}} = S_{DN}} & (N)\end{matrix}$

wherein each of (S_(D1)) to (S_(DN)) is a signal detected at eachdetection temperature; each of (S_(T1D1)) to (S_(TNDN)) is a variablerepresenting a signal of interest generated by each signal-generatingmeans at each detection temperature; and the total number of thevariables is N².

In the above mathematical equations, “ . . . ” in a horizontal dot linerepresents a variable or variables of which number is dependent on thenumber of N. For instance, where N is three, there is no additionalvariable represented by the horizontal dot line. Where N is four, thereis an additional variable in the number of (N−3) represented by thehorizontal dot line. Furthermore, “ . . . ” in a vertical dot linerepresents an equation or equations of which number is dependent on thenumber of N. For instance, where N is three, there is no additionalequation represented by the vertical dot line. Where N is four, there isan additional equation in the number of (N−3) represented by thevertical dot line.

Where N is two, the equation set is presented as follows:S _(T1D1) +S _(T2D1) =S _(D1)  (1)S _(T1D2) +S _(T2D2) =S _(D2)  (2)

(S_(D1)), the signal detected at the first detection temperature (D1)consists of signals of interest in the number of N generated by thesignal-generating means in the number of N for the target nucleic acidsequences in the number of N at the first detection temperature (D1). Inother words, (S_(D1)), the signal detected at the first detectiontemperature consists of (S_(T1D1)), the signal of interest generated bythe first signal-generating means for the first target nucleic acidsequence at the first detection temperature to (S_(TND1)), the signal ofinterest generated by the N^(th) signal-generating means for the N^(th)target nucleic acid sequence at the first detection temperature.

The equations for other detection temperatures may be interpreted asdescribed above for the first detection temperature.

For example, (S_(DN)), the signal detected at the N^(th) detectiontemperature (DN) consists of N

signals of interest generated by the signal-generating means in thenumber of N for the target nucleic acid sequences in the number of N atthe N^(th) detection temperature (DN). (S_(DN)), the signal detected atthe N^(th) detection temperature consists of (S_(T1DN)), the signal ofinterest generated by the first signal-generating means for the firsttarget nucleic acid sequence at the N^(th) detection temperature to(S_(TNDN)), the signal of interest generated by the N^(th)signal-generating means for the N^(th) target nucleic acid sequence atthe N^(th) detection temperature.

(S_(D1)) to (S_(DN)) may be a measured signal by detectors, i.e., aconstant value. (S_(T1D1)) to (S_(TNDN)) are unknown variables to beobtained. The present invention is drawn to methods for obtainingsolutions to at least one of the variables (S_(T1D1)) to (S_(TNDN)) byusing measured signals (S_(D1)) to (S_(DN)).

In a particular example, where the target nucleic acid sequences in thenumber of N in a sample comprises T1, T2 and T3, and the detectiontemperatures in the number of N comprises 60° C., 72° C. and 95° C., thefollowing equations in the number of N each of which comprises variablesrepresenting the signals of interest generated at each detectiontemperature for the target nucleic acid sequences may be provided:S _(T1D1) +S _(T2D1) +S _(T3D1) =S _(D1)S _(T1D2) +S _(T2D2) +S _(T3D2) =S _(D2)S _(T1D3) +S _(T2D3) +S _(T3D3) =S _(D3)Step (c): Establishing Additional Equations

Afterwards, provided are additional equations in the number of (N²−N)each of which comprises at least one variable selected from the groupconsisting of the variables, (S_(T1D1)) to (S_(TNDN)).

Following setting up the equations (1) to (N), additional equations inthe number of (N²−N) are established such that the additional equationsare provided for obtaining solutions to at least one of the variables(particularly at least two of the variables, more particularly at leastthree of the variables, still more particularly at least four of thevariables, still much more particularly at least five of the variables)in the equations (1) to (N) by a mathematical processing.

Mathematically, the equations in the number of N² provided in the steps(b) and (c) may provide solutions to at least one of the variables.

For instance, where the additional equations enable that the equations(1) to (N) have the same variable set (i.e., the equations (1) to (N)are converted to a simultaneous equation), the solution to at least oneof the variables in the equations (1) to (N) can be obtained by solvingthe simultaneous equation (particularly, linear simultaneous equationwith variables in the number of N). Alternatively, the solution to atleast one of the variables in the equations (1) to (N) may be obtainedin a different manner from the simultaneous equation-solving approach.

According to an embodiment, the additional equations in the number of(N²−N) are selected from the group of the following equations:f(S _(TjDα) ,S _(TjDβ))=RV_(Tj(DαDβ))  (X), andS _(TjDK)=an arbitrarily selected value  (XI)

wherein, j in Tj represents all integers starting from 1 to a N^(th)integer; for each j^(th) target nucleic acid, α and β jointly representa combination of two integers selected from 1 to N; wherein the numberof the combination is represented by _(N)C₂; for each j^(th) targetnucleic acid, K is at least one of integers selected from 1 to N;RV_(Tj(DαDβ)) is a reference value (RV) of the j^(th) target nucleicacid sequence (Tj) representing relationship reflecting change insignals provided by the j^(th) signal-generating means at the α^(th)detection temperature and the β^(th) detection temperature; f(S_(TjDα),S_(TjDβ)) represents a function of S_(TjDα) and S_(TjDβ);

wherein S_(TjDK)=an arbitrarily selected value in the equation (XI) maybe selected with a proviso that the j^(th) signal-generating means isprepared to generate no signal in the presence of the j^(th) targetnucleic acid sequence at the K^(th) detection temperature; and

wherein when the equation (XI) is selected as the additional equations,the other additional equations are selected from the equation (X) inwhich α or β for the j^(th) target nucleic acid sequence is differentfrom K for the j^(th) target nucleic acid sequence.

According to an embodiment, it is convenient in terms of performance ofthe present invention that zero (0) may be used as S_(TjDK)(S_(TjDK)=0). Alternatively, either negative numeric constant orpositive numeric constant may be used as S_(TjDK) (S_(TjDK)=negativenumeric constant or S_(TjDK)=positive numeric constant).

S_(TjDα) and S_(TjDβ) represents signals provided by the j^(th)signal-generating means at the α^(th) detection temperature (Dα) and atthe β^(th) detection temperature (Dβ), respectively, when the j^(th)target nucleic acid sequence (Tj) is present.

The equation (X) is presented for describing a plurality of equations asa single general equation.

In the mathematical equation (X), j in Tj represents all integersstarting from 1 to a N^(th) integer, and α and β jointly represent acombination of two integers selected from 1 to N. The term “combination”in conjunction with α and β is a mathematic combination meaning a way ofselecting items from a collection, such that the order of selection doesnot matter. The combination of α and β means a combination of two amongthe detection temperatures in the number of N. The number of thecombination of α and β is represented by _(N)C₂.

According to an embodiment, where the target nucleic acid sequences arein the number of N, the equation (X) permits to describe equations inthe number of “N× [_(N)C₂]”.

For instance, where N is 3, j comprises 1, 2 and 3 and the combinationof α and β comprises (1, 2), (1, 3) and (2, 3). In this case, theequation (X) describes nine (9) equations.

For example, “f(S_(TjDα), S_(TjDβ))=RV_(Tj(DαDβ))” may comprise:

(i) in the case of j=1;f(S _(T1D1) ,S _(T1D2))=RV_(T1(D1D2)),f(S _(T1D1) ,S _(T1D3))=RV_(T1(D1D3)),f(S _(T1D2) ,S _(T1D3))=RV_(T1(D2D3)),

(ii) in the case of j=2;f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2)),f(S _(T2D1) ,S _(T2D3))=RV_(T2(D1D3)),f(S _(T2D2) ,S _(T2D3))=RV_(T2(D2D3)),

(ii) in the case of j=3f(S _(T3D1) ,S _(T3D2))=RV_(T3(D1D2)),f(S _(T3D1) ,S _(T3D3))=RV_(T3(D1D3)), andf(S _(T3D2) ,S _(T3D3))=RV_(T3(D2D3)).

According to an embodiment, where the additional equations in the numberof (N²−N) are selected from the equation (X), they may be selected suchthat equations in the number of (N−1) are selected from the equationgroup for each j.

More particularly, such (N−1) equation-selection may be carried out suchthat the selected (N−1) equations for each j provides a simultaneousequation with variables in the number of N to obtain solutions to atleast one of the variables by the equations (1) to (N).

In the particular example in which N is 3 and signals at the detectiontemperature D1 are analyzed, two equations (i.e. N−1=3−1=2) for each jmay be selected among three presentable equations. Six (6) additionalequations selected from the nine (9) equations described above areselected as follows:f(S _(T1D1) ,S _(T1D2))=RV_(T1(D1D2))f(S _(T1D1) ,S _(T1D3))=RV_(T1(D1D3))f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2))f(S _(T2D1) ,S _(T2D3))=RV_(T2(D1D3))f(S _(T3D1) ,S _(T3D2))=RV_(T3(D1D2))f(S _(T3D1) ,S _(T3D3))=RV_(T3(D1D3))

Particularly, the additional equations in the number of (N²−N) compriseequations selected from an equation group represented by the equation(X).

According to an embodiment, a portion of the additional equations in thenumber of (N²−N) can be selected from the group of the followingequation:S _(TjDK)=an arbitrarily selected value  (XI)

Where the j^(th) signal-generating means is prepared to generate nosignal in the presence of the j^(th) target nucleic acid sequence at theK^(th) detection temperature, an equation to allow the S_(TjDK) to havean arbitrarily selected value.

According to an embodiment, signals may not be generated at a pluralityof detection temperatures by adjusting characteristics of the j^(th)signal-generating means.

The equation (XI) is presented for describing a plurality of equationsas a single general equation.

Where a detection temperature is selected to generate no signal for eachof the target nucleic acid sequences in the number of N, an equationallowing signal for a target nucleic acid sequence at the detectiontemperature to have an arbitrarily selected value. The equation may berepresented by the equation (XI).

For instance, where the target nucleic acid sequences are in the numberof N (i.e., N=3), a first signal-generating means for a first targetnucleic acid sequence is designed to generate no signal at a seconddetection temperature and a third detection temperature and a secondsignal-generating means for a second target nucleic acid sequence isdesigned to generate no signal at a third detection temperature, theequation (XI) “S_(TjDK)=an arbitrarily selected value” comprisesS_(T1D2)=an arbitrarily selected value, S_(T1D3)=an arbitrarily selectedvalue and S_(T2D3)=an arbitrarily selected value. All or a portion ofthe three equations may be selected.

According to an embodiment, the additional equations comprise anequation or equations selected from an equation group represented by theequation (X) as well as an equation or equations selected from anequation group represented by the equation (XI).

The above-described embodiment describes two approaches to obtain thesolution to at least one of the variables in the equations (1) to (N).

The first approach is to use the equation (X) as the additionalequations in the number of (N²−N).

In the equation (X), RV_(Tj(DαDβ)) as a reference value is apredetermined value.

The reference value is obtained through a mathematical processing ofsignals provided by a signal-generating means at two detectiontemperatures and the second detection temperature. Such mathematicalprocessing is a function of the signals. The function used in obtainingreference values include any function so long as it gives a relationshipof change in signals provided by the signal-generating means at the twodetection temperatures. For instance, the function may be presented as amathematical processing such as addition, multiplication, subtractionand division of signals.

As described above, because RV_(Tj(DαDβ)) as a reference value is apredetermined value, RV_(Tj(DαDβ)) may serve as a convertor for (i)converting the variables (S_(TjD1)) to (S_(TjDN)) in the equations (1)to (N) into a variable selected from the variables (S_(TjD1)) to(S_(TjDN)).

According to an embodiment, the equation (X) comprising the referencevalue (RV) of the j^(th) target nucleic acid sequence (Tj) is used toconvert the variables (S_(TjD1)) to (S_(TjDN)) in the equations (1) to(N) into a variable selected from the variables (S_(TjD1)) to(S_(TjDN)).

The reference values, RV_(Tj(DαDβ)) used in this invention may beobtained in various manners. For instance, the reference value may begiven as an anticipated value. In considering a target sequence, asignal-generating means and detection temperatures, the reference valuerepresenting a relationship of change in signals at the detectiontemperatures may be anticipated. Alternatively, the reference value maybe given as an experimental or practical value by performing experimentsfor obtaining the reference value.

According to an embodiment, RV_(Tj(DαDβ)) is obtained by (i-1)incubating the j^(th) target nucleic acid sequence with the j^(th)signal-generating means for detection of the j^(th) target nucleic acidsequence, (i-2) detecting signals at the α^(th) detection temperatureand the β^(th) detection temperature, and (i-3) then obtaining adifference between the signals detected at the α^(th) detectiontemperature and the β^(th) detection temperature.

The term “difference between signals detected at the α^(th) detectiontemperature and the β^(th) detection temperature” is an embodiment of arelationship of change in signals at the α^(th) detection temperatureand the β^(th) detection temperature.

According to an embodiment, the difference between the signals detectedat the α^(th) detection temperature and the β^(th) detection temperaturecomprises a difference to be obtained by mathematically processing thesignal detected at the α^(th) detection temperature and the β^(th)detection temperature.

According to an embodiment, where the mathematical processing is done,the characteristics of the signal should be vulnerable to themathematical processing. In certain embodiment, the mathematicalprocessing includes calculation (e.g., addition, multiplication,subtraction and division) using signals or obtaining other valuesderived from signals.

The difference between the signals at the α^(th) detection temperatureand the β^(th) detection temperature may be expressed as a numericalvalue or plot with signal characteristics.

The mathematical processing of the signals for obtaining the differencemay be carried out by various calculation methods and theirmodifications.

In particular, the mathematical processing of the signals for obtainingthe difference may be carried out by calculating a ratio between signalsat the α^(th) detection temperature and the β^(th) detectiontemperature.

For instance, the ratio of the end-point intensity of the signaldetected at the β^(th) detection temperature to the end-point intensityof the signal detected at the α^(th) detection temperature may be usedas reference values.

The mathematical processing for obtaining the difference may be carriedout in various fashions. The mathematical processing may be carried outby use of a machine. For example, the signals may be undergone amathematical processing by a processor in a detector or real-time PCRdevice. Alternatively, the signals may be manually undergone amathematical processing particularly according to a predeterminedalgorithm.

According to an embodiment of the present invention, RVs for the targetnucleic acid sequences in the number of N at detection temperatures inthe number of N are different from each other.

Particularly, reference values for different target nucleic acidsequences calculated by using signals between the same two-detectiontemperatures are different. For example, where there are three targetnucleic acid sequences, RV_(T1(DαDβ)), RV_(T2(DαDβ)) and RV_(T3(DαDβ))may be different from each other. According to an embodiment, signalgenerating means are used to provide different reference values fordifferent target nucleic acid sequences.

Where the RV values are different from each other, a quantitativeexpression describing a difference extent may be varied depending onapproaches for calculating the RV values.

According to an embodiment, signals for obtaining reference values maybe processed by a common calculation method to provide a reference valuefor comparison, and then a difference extent between two referencevalues may be obtained by using the reference value for comparison.According to an embodiment, the common calculation method is division oftwo signals.

For instance, while two signals are processed by subtraction forobtaining reference values used to analyze signals according to thepresent method, the two signals may be processed by division forobtaining a reference value for comparison.

According to an embodiment, one reference value is at least 1.1-fold,1.2-fold, 1.3-fold, 1.5-fold, 1.7-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, 4-fold, 5-fold or 10-fold larger than the other referencevalue.

According to an embodiment, some of reference values used in the presentmethod may be the same to one another.

According to an embodiment of this invention, the signal-generatingmeans for the reference value may be the same as those for the detectionof the target nucleic acid sequences.

According to an embodiment, the incubation conditions for obtaining thereference values are the same as those for the sample.

For a target nucleic acid sequence, the reference values may be obtainedunder various reaction conditions including the amounts of components(e.g. the target nucleic acid sequence, signal-generating means, enzymesand dNTPs), buffer pH or reaction time. According to an embodiment ofthis invention, the reference value may be obtained under reactionconditions sufficient to provide a saturated signal at the reactioncompletion. According to an embodiment of this invention, the differencebetween the signals obtained in obtaining the reference value has acertain range and the reference value is selected within the certainrange or with referring to the certain range. According to an embodimentof this invention, the reference value may be selected with maximum orminimum value of the certain range or with referring to maximum orminimum value of the certain range. Particularly, the reference valuemay be modified in considering standard variation of the referencevalues obtained in various conditions, acceptable error ranges,specificity or sensitivity.

f(S_(TjDα), S_(TjDβ)) in “f(S_(TjDα), S_(TjDβ))=RV_(Tj(DαDβ))” may bepresented by a mathematical expression for calculating RV_(Tj(DαDβ)).

According to an embodiment, f(S_(TjDα), S_(TjDβ))=RV_(Tj(DαDβ))comprises S_(TjDα)/S_(TjDβ)=RV_(TjDα/Dβ) (XII) orS_(TjDβ)/S_(TjDα)=RV_(TjDβ/Dα) (XIII).

Where RV is obtained by using a ratio between signals at the α^(th)detection temperature and the β^(th) detection temperature, f(S_(TjDα),S_(TjDβ)) in “f(S_(TjDα), S_(TjDβ))=RV_(Tj(DαDβ))” may be presented by amathematical expression for calculating ratio between S_(TjDα) andS_(TjDβ). In the term “RV_(Tj(DαDβ))”, Tj(DαDβ) indicates that acalculated RV corresponds to the reference value for the j^(th) targetnucleic acid sequence obtained by calculating a difference betweensignals at the α^(th) detection temperature (Dα) and the β^(th)detection temperature (Dβ).

Upon determining a method for calculation RV, the term “Tj(DαDβ)” may bespecifically expressed in considering the calculation method. Forexample, the term “RV_(TjDα/Dβ” means a reference value for the j) ^(th)target nucleic acid sequence obtained by calculating a ratio of a signaldetected at the α^(th) detection temperature to a signal detected at theβ^(th) detection temperature.

According to an embodiment, RV_(TjDα/Dβ) is obtained by (i-1) incubatingthe j^(th) target nucleic acid sequence with the j^(th)signal-generating means for detection of the j^(th) target nucleic acidsequence, (i-2) detecting signals at the α^(th) detection temperatureand the β^(th) detection temperature, and (i-3) then calculating a ratioof a signal detected at the α^(th) detection temperature to a signaldetected at the β^(th) detection temperature; RV_(TjDβ/Dα) is obtainedby performing the steps (i-1) and (i-2) and then calculating a ratio ofthe signal detected at the β^(th) detection temperature to the signaldetected at the α^(th) detection temperature.

According to an embodiment, the additional equations in the number ofN²−N are selected from the groups of the following equations:S _(TjDα) /S _(TjDβ)=RV_(TjDα/Dβ)  (XII)S _(TjDβ) /S _(TjDα)=RV_(TjDβ/Dα)  (XIII)

wherein the equation for each j^(th) target nucleic acid at the α^(th)detection temperature and the β^(th) detection temperature is selectedfrom the equations (XII) and (XIII). Particularly, the equation for eachj^(th) target nucleic acid at the α^(th) detection temperature and theβ^(th) detection temperature is selected from one of the equations (XII)and (XIII).

The additional equation(s) selected from an equation group representedby the equation (X) together with the equations (1) to (N) are used forobtaining solutions to at least one of the variables.

In the particular example (N=3), the equation (XII) or (XIII) ispresented as follows:

(i) in the case of j=1;f(S _(T1D1) ,S _(T1D2))=RV_(T1(D1D2)) may comprise:S _(T1D1) /S _(T1D2)=RV_(T1D1/D2) or S _(T1D2) /S _(T1D1)=RV_(T1D2/D1)f(S _(T1D1) ,S _(T1D3))=RV_(T1(D1D3)) may comprise:S _(T1D1) /S _(T1D3)=RV_(T1D1/D3) or S _(T1D3) /S _(T1D1)=RV_(T1D3/D1)f(S _(T1D2) ,S _(T1D3))=RV_(T1(D2D3)) may comprise:S _(T1D2) /S _(T1D3)=RV_(T1D2/D3) or S _(T1D3) /S _(T1D2)=RV_(T1D3/D2)

(ii) in the case of j=2;f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2)) may comprise:S _(T2D1) /S _(T2D2)=RV_(T2D1/D2) or S _(T2D2) /S _(T2D1)=RV_(T2D2/D1)f(S _(T2D1) ,S _(T2D3))=RV_(T2(D1D3)) may comprise:S _(T2D1) /S _(T1D3)=RV_(T2D1/D3) or S _(T2D3) /S _(T2D1)=RV_(T2D3/D1)f(S _(T2D2) ,S _(T2D3))=RV_(T2(D2D3)) may comprise:S _(T2D2) /S _(T2D3)=RV_(T2D2/D3) or S _(T2D3) /S _(T2D2)=RV_(T2D3/D2)

(ii) in the case of j=3f(S _(T3D1) ,S _(T3D2))=RV_(T3(D1D2)) may comprise:S _(T3D1) /S _(T3D2)=RV_(T3D1/D2) or S _(T3D2) /S _(T3D1)=RV_(T3D2/D1)f(S _(T3D1) ,S _(T3D3))=RV_(T3(D1D3)) may comprise:S _(T3D1) /S _(T3D3)=RV_(T3D1/D3) or S _(T3D3) /S _(T3D1)=RV_(T3D3/D1)f(S _(T3D2) ,S _(T3D3))=RV_(T3(D2D3)) may comprise:S _(T3D2) /S _(T3D3)=RV_(T3D2/D3) or S _(T3D3) /S _(T3D2)=RV_(T3D3/D2)

According to an embodiment, a plurality of equations for calculating RVfor a certain target nucleic acid sequence at selected two detectiontemperatures may be presented. According to an embodiment, in such case,a single equation among a plurality of equations is selected. Forexample, where j=1 (i.e., T1) and two detection temperatures are 60° C.(D1) and 72° C. (D2), “f(S_(T1D1), S_(T1D2))=RV_(T1(D1D2))” may compriseS_(T1D1)/S_(T1D2)=RV_(T1D1/D2) and S_(T1D2)/S_(T1D1)=RV_(T1D2/D1). Asingle equation among the two particular equations is used to calculateRV for the target nucleic acid sequence (T1).

Where signals at the detection temperature D1 are analyzed, six (6)additional equations selected from the equations described above may beselected as follows:S _(T1D1) /S _(T1D2)=RV_(T1D1/D2) (or S _(T1D2) /S _(T1D1)=RV_(T1D2/D1))S _(T1D1) /S _(T1D3)=RV_(T1D1/D3) (or S _(T1D3) /S _(T1D1)=RV_(T1D3/D1))S _(T2D1) /S _(T2D2)=RV_(T2D1/D2) (or S _(T2D2) /S _(T2D1)=RV_(T2D2/D1))S _(T2D1) /S _(T2D3)=RV_(T2D1/D3) (or S _(T2D3) /S _(T2D1)=RV_(T2D3/D1))S _(T3D1) /S _(T3D2)=RV_(T3D1/D2) (or S _(T3D2) /S _(T3D1)=RV_(T3D2/D1))S _(T3D1) /S _(T3D3)=RV_(T3D1/D3) (or S _(T3D3) /S _(T3D1)=RV_(T3D3/D1))

Finally, the equation set for obtaining solutions to variables mayinclude the following:S _(T1D1) +S _(T2D1) +S _(T3D1) =S _(D1)S _(T1D2) +S _(T2D2) +S _(T3D2) =S _(D2)S _(T1D3) +S _(T2D3) +S _(T3D3) =S _(D3)S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)S _(T1D1) /S _(T1D3)=RV_(T1D1/D3)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)S _(T2D1) /S _(T2D3)=RV_(T2D1/D3)S _(T3D1) /S _(T3D2)=RV_(T3D1/D2)S _(T3D1) /S _(T3D3)=RV_(T3D1/D3)

Because RV_(T1D1/D2), RV_(T1D1/D3), RV_(T2D1/D2), RV_(T2D1/D3),RV_(T3D1/D2) and RV_(T3D1/D3) are an experimentally predetermined value,the solutions to the variables (S_(T1D1)), (S_(T2D1)), (S_(T3D1)),(S_(T1D2)), (S_(T2D2)), (S_(T3D2)), (S_(T1D3)), (S_(T2D3)) and(S_(T3D3)) may be obtained by the nine (9) equations.

The second approach to obtain the solution to at least one of thevariables in the equations (1) to (N) is to use the equations (X) and(XI) as the additional equations in the number of (N²−N). S_(TjDK)=anarbitrarily selected value in the equation (XI) may be selected with aproviso that the j^(th) signal-generating means is prepared to generatesubstantially no signal in the presence of the j^(th) target nucleicacid sequence at the K^(th) detection temperature.

According to an embodiment, when the equation (XI) is selected as theadditional equations, the other additional equations are selected fromthe equation (X) in which α or β for the j^(th) target nucleic acidsequence is different from K for the j^(th) target nucleic acidsequence.

In other words, when at least one equation is selected from an equationgroup represented by the equation (XI) as the additional equations, aportion of an equation group represented by the equation (X) in which αor β for the j^(th) target nucleic acid sequence is the same as K forthe j^(th) target nucleic acid sequence is excluded from the otheradditional equations and the other additional equations are selectedfrom the equation (X) except for the exclusion.

Therefore, the phrase used herein “when the equation (XI) is selected asthe additional equations, the other additional equations are selectedfrom the equation (X) in which α or β for the j^(th) target nucleic acidsequence is different from K for the j^(th) target nucleic acidsequence” may be also described as “when the equation (XI) is selectedas the additional equations, the other additional equations are selectedfrom the equation (X) except for an equation in which α or β for thej^(th) target nucleic acid sequence is the same as K for the j^(th)target nucleic acid sequence.

In a particular example in which three target nucleic acid sequences areanalyzed (i.e. N=3 and j comprises 1, 2 and 3), a signal generatingmeans for a target nucleic acid sequence (T1) is prepared to generatesubstantially no signal even in the presence of T1 at the 72° C. (D2)and 95° C. (D3) detection temperatures and a signal generating means fora target nucleic acid sequence (T2) is prepared to generatesubstantially no signal even in the presence of T2 at the 95° C.detection temperature (D3) (see FIG. 3A), the equation (XI) may comprisethe following equations:S _(T1D2)=0 (j=1 and K=2),S _(T1D3)=0 (j=1 and K=3), andS _(T2D3)=0 (j=2 and K=3).

Meanwhile, the equation (X) “f(S_(TjDα), S_(TjDβ))=RV_(Tj(DαDβ))” maycomprise:

(i) in the case of j=1;f(S _(T1D1) ,S _(T1D2))=RV_(T1(D1D2)),f(S _(T1D1) ,S _(T1D3))=RV_(T1(D1D3)),f(S _(T1D2) ,S _(T1D3))=RV_(T1(D2D3)),

(ii) in the case of j=2;f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2)),f(S _(T2D1) ,S _(T2D3))=RV_(T2(D1D3)),f(S _(T2D2) ,S _(T2D3))=RV_(T2(D2D3)),

(ii) in the case of j=3f(S _(T3D1) ,S _(T3D2))=RV_(T3(D1D2)),f(S _(T3D1) ,S _(T3D3))=RV_(T3(D1D3)), andf(S _(T3D2) ,S _(T3D3))=RV_(T3(D2D3)).

Where “S_(T2D3)=0 (j=2 and K=3)” is used as one of the additionalequation, the other additional equations may comprise two equationsamong the equation (X) (j=1), two equations among the equation (X) (j=3)and one equation among the equation (X) (j=2). In a certain embodiment,for such equation selection, equations in which α or β for the targetnucleic acid sequence (j=2, i.e. T2) is the same as K (i.e., 3) for thetarget nucleic acid sequence (j=2, i.e. T2) are excluded. In otherwords, equations containing the D3 detection temperature among“f(S_(T2Dα), S_(T2Dβ)) RV_(T2(DαDβ))” are excluded.

Where signals at the detection temperature 60° C. (D1) are analyzed,equations comprising variables containing D1 may be selected as follows:S _(T2D3)=0f(S _(T1D1) ,S _(T1D2))=RV_(T1(D1D2))f(S _(T1D1) ,S _(T1D3))=RV_(T1(D1D3))f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2))f(S _(T3D1) ,S _(T3D2))=RV_(T3(D1D2))f(S _(T3D1) ,S _(T3D3))=RV_(T3(D1D3))

Instead of using “S_(T2D3)=0 (j=2 and K=3)”, S_(T1D2)=0 (j=1 and K=2)and S_(T1D3)=0 (j=1 and K=3) may be used as two of the additionalequations. The other additional equations may comprise two equationsamong the equation (X) (j=2) and two equations among the equation (X)(j=3). In a certain embodiment, for such equation selection, equationsin which α or β for the target nucleic acid sequence (j=1, i.e. T1) isthe same as K (i.e., 2 or 3) for the target nucleic acid sequence (j=1,i.e. T1) are excluded. In other words, equations containing D2 or D3 asthe detection temperature among “f(S_(T1Dα), S_(T1Dβ))=RV_(T1(DαDβ))”are excluded. Where signals at the detection temperature 60° C. (D1) areanalyzed, equations comprising variables containing D1 may be selectedas follows:S _(T1D2)=0S _(T1D3)=0f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2))f(S _(T2D1) ,S _(T2D3))=RV_(T2(D1D3))f(S _(T3D1) ,S _(T3D2))=RV_(T3(D1D2))f(S _(T3D1) ,S _(T3D3))=RV_(T3(D1D3))

According to an embodiment, the additional equations in numbers of N²−Nare selected from the groups of the following equations:S _(TjDα) /S _(TjDβ)=RV_(TjDα/Dβ)  (XII)S _(TjDβ) /S _(TjDα)=RV_(TjDβ/Dα)  (XIII)S _(TjDK)=an arbitrarily selected value  (XI)

wherein the equation for each j^(th) target nucleic acid at the α^(th)detection temperature and the β^(th) detection temperature is selectedfrom the equations (XII) and (XIII);

wherein when the equation (XI) is selected as the additional equations,the other additional equations are selected from the equations (XII) and(XIII) in which α or β for the j^(th) target nucleic acid sequence isdifferent from K for the j^(th) target nucleic acid sequence.

In a particular example in which three target nucleic acid sequences areanalyzed (i.e. N=3 and j comprises 1, 2 and 3), a signal generatingmeans for a target nucleic acid sequence (T1) is prepared to generatesubstantially no signal even in the presence of T1 at the 72° C. (D2)and 95° C. (D3) detection temperatures and a signal generating means fora target nucleic acid sequence (T2) is prepared to generatesubstantially no signal even in the presence of T2 at the 95° C.detection temperature (D3) (see FIG. 3A), there are various equationsets available for obtaining solutions to variables.

Where “S_(T2D3)=0 (j=2 and K=3)” is used as one of the additionalequation, one of the equation sets for obtaining solutions to variablesmay include the following:S _(T1D1) +S _(T2D1) +S _(T3D1) =S _(D1)S _(T1D2) +S _(T2D2) +S _(T3D2) =S _(D2)S _(T1D3) +S _(T2D3) +S _(T3D3) =S _(D3)S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)S _(T1D1) /S _(T1D3)=RV_(T1D1/D3)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)S _(T2D3)=0S _(T3D1) /S _(T3D2)=RV_(T3D1/D2)S _(T3D1) /S _(T3D3)=RV_(T3D1/D3)

Because RV_(T1D1/D2), RV_(T1D1/D3), RV_(T2D1/D2), RV_(T3D1/D2) andRV_(T3D1/D3) are constants determined experimentally or arbitrarily, thesolutions to the variables (S_(T1D1)), (S_(T2D1)), (S_(T3D1)),(S_(T1D2)), (S_(T2D2)), (S_(T3D2)), (S_(T1D3)) and (S_(T3D3)) may beobtained by the nine (9) equations.

Instead of using “S_(T2D3)=0 (j=2 and K=3)”, S_(T1D2)=0 (j=1 and K=2)and S_(T1D3)=0 (j=1 and K=3) may be used as two of the additionalequations. One of the equation sets for obtaining solutions to variablesmay include the following:S _(T1D1) +S _(T2D1) +S _(T3D1) =S _(D1)S _(T1D2) +S _(T2D2) +S _(T3D2) =S _(D2)S _(T1D3) +S _(T2D3) +S _(T3D3) =S _(D3)S _(T1D2)=0S _(T1D3)=0S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)S _(T2D1) /S _(T2D3)=RV_(T2D1/D3)S _(T3D1) /S _(T3D2)=RV_(T3D1/D2)S _(T3D1) /S _(T3D3)=RV_(T3D1/D3)Because RV_(T2D1/D2), RV_(T2D1/D3), RV_(T3D1/D2) and RV_(T3D1/D3) areconstants determined experimentally or arbitrarily, the solutions to thevariables (S_(T1D1)), (S_(T2D1)), (S_(T3D1)), (S_(T2D2)), (S_(T3D2)),(S_(T2D3)) and (S_(T3D3)) may be obtained by the nine (9) equations.

According to an embodiment, even though the j^(th) signal-generatingmeans is prepared to generate no signal in the presence of the j^(th)target nucleic acid sequence at the K^(th) detection temperature, theequation “S_(TjDK)=an arbitrarily selected value” may not be used as anadditional equation. In such case, RV_(Tj(DαDβ)) in which α or β is thesame as K may be an experimentally-obtained value.

According to an embodiment, when the j^(th) signal-generating means isprepared to generate no signal in the presence of the j^(th) targetnucleic acid sequence at the K^(th) detection temperature, RV_(Tj(DαDβ))in which α or β is the same as K is an arbitrarily selected value.

Particularly, when f(S_(TjDα), S_(TjDβ)) in the equation (X) ispresented by a mathematical expression for calculating ratio betweensignals at the α^(th) detection temperature (Dα) and the β^(th)detection temperature (Dβ), RV_(Tj(DαDβ)) may be selected as anarbitrarily selected value.

Particularly, the arbitrarily selected value as RV_(Tj(DαDβ)) may beselected such that the solution of S_(TjDK) is rendered to be a value oraround the value (e.g. “0” or around “0”) in solving procedures of anequation set.

According to an embodiment, the arbitrarily selected value asRV_(Tj(DαDβ)) may be the same as, greater or less than RV_(Tj(DαDβ))calculated with practically-obtained signal values.

Practically, even when the j^(th) signal-generating means is prepared togenerate no signal in the presence of the j^(th) target nucleic acidsequence at the K^(th) detection temperature, it is usually to generatesignals with very weak intensities (e.g., background signal). Therefore,RV_(Tj(DαDβ)) in which α or β is the same as K may be obtainedexperimentally.

It is very interesting that the present method allows for quantificationof a signal amount (e.g. signal intensity) generated by each of thesignal-generating means. As described above, the solutions to thevariables (S_(T1D1)) to (S_(TNDN)) permit quantification of a signalamount (e.g. signal intensity) generated by each of thesignal-generating means, which may be applied to quantification of thetarget nucleic acid sequences.

Step (d): Obtaining Solutions to Variables

Finally, the solutions to at least one of the variables by the equationsin the number of N² provided in the steps (b) and (c) fordifferentiating at least one of the signals of interest to be assignedto at least one of the target nucleic acid sequences in the number of N.

As described above, the equations in the number of N² provided in thesteps (b) and (c) are used to obtain the solutions to at least one ofthe variables for differentiating at least one of the signals ofinterest to be assigned to at least one of the target nucleic acidsequences in the number of N, whereby the presence of at least one ofthe target nucleic acid sequences in the number of N may be determined.

Particularly, the solutions to at least one (e.g., at least two, atleast three, at least four, at least five, at least six, at least nine,at least sixteen, at least twenty-five or at least N²) of the variablesare obtained by using the equations in the number of N² provided in thesteps (b) and (c) for differentiating at least one (e.g., at least two,at least three, at least four, at least five, at least six, at leastnine, at least sixteen, at least twenty-five or at least N²) of thesignals of interest to be assigned to at least one (e.g., at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine and at least ten or at least N) ofthe target nucleic acid sequences.

According to an embodiment, the present method is used to obtainsolutions (values) to the variables in the number of N each of which isselected from a variable group for each target nucleic acid sequence.

According to an embodiment, depending on approaches for generatingsignals, a threshold value may be employed to analyze whether theobtained solutions to the variables may be significant. A negativecontrol, sensitivity or label used may be considered for determining thethreshold value. According to an embodiment of this invention, athreshold value may be determined by user or automatically.

According to an embodiment, the threshold value is determined withreferring to signals detected using only one of the target nucleic acidsequences at a detection temperature.

According to an embodiment, the threshold value may be used specificallyto each target nucleic acid sequence or each detection temperature.

According to an embodiment, where signals are generated in a real-timemanner associated with target amplification by PCR, the signals at eachamplification cycle or some selected cycles are mathematically processedwith the reference values and the calculation results are plottedagainst cycles and used for differentiating the signals of interest(e.g., determination of the presence of the target nucleic acidsequence).

According to an embodiment, a threshold value is applied to the plottingresult to determine the significance of the signal of interest.

According to an embodiment of this invention, the target nucleic acidsequences comprises a nucleotide variation.

The term “nucleotide variation” used herein refers to any single ormultiple nucleotide substitutions, deletions or insertions in a DNAsequence at a particular location among contiguous DNA segments that areotherwise similar in sequence. Such contiguous DNA segments include agene or any other portion of a chromosome. These nucleotide variationsmay be mutant or polymorphic allele variations. For example, thenucleotide variation detected in the present invention includes SNP(single nucleotide polymorphism), mutation, deletion, insertion,substitution and translocation. Exemplified nucleotide variationincludes numerous variations in a human genome (e.g., variations in theMTHFR (methylenetetrahydrofolate reductase) gene), variations involvedin drug resistance of pathogens and tumorigenesis-causing variations.The term nucleotide variation used herein includes any variation at aparticular location in a nucleic acid sequence. In other words, the termnucleotide variation includes a wild type and its any mutant type at aparticular location in a nucleic acid sequence.

According to an embodiment of this invention, the nucleotide variationdetected by the present invention is a SNP (single nucleotidepolymorphism).

III. Kits for Differentiating Signals of Interest for Target NucleicAcid Sequences

In still another aspect of this invention, there is provided a kit fordifferentiating signals of interest for each of two target nucleic acidsequences comprising a first target nucleic acid sequence (T1) and asecond target nucleic acid sequence (T2) in a sample, which are notdifferentiable by a single type of detector, comprising:

(a) a first signal-generating means for detection of the first targetnucleic acid sequence (T1) and a second signal-generating means fordetection of the second target nucleic acid sequence (T2) twosignal-generating means for detection of the two target nucleic acidsequences; and

(b) an instruction that describes the present method of the Aspect Ititled as Differentiation of Signals of Interest for Two Target NucleicAcid Sequences.

In further aspect of this invention, there is provided a kit fordifferentiating signals of interest for each of target nucleic acidsequences in the number of N in a sample, which are not differentiableby a single type of detector, comprising:

(a) signal-generating means in the number of N for detection of thetarget nucleic acid sequences in the number of N; and

(b) an instruction that describes the present method of the Aspect IItitled as Differentiation of Signals of Interest for at least ThreeTarget Nucleic Acid Sequences.

Since the kits of this invention are prepared to perform the presentmethods, the common descriptions between them are omitted in order toavoid undue redundancy leading to the complexity of this specification.

All of the present kits described hereinabove may optionally include thereagents required for performing target amplification reactions (e.g.,PCR reactions) such as buffers, DNA polymerase cofactors, anddeoxyribonucleotide-5-triphosphates. Optionally, the kits may alsoinclude various polynucleotide molecules, reverse transcriptase, variousbuffers and reagents, and antibodies that inhibit DNA polymeraseactivity. The kits may also include reagents necessary for performingpositive and negative control reactions. Optimal amounts of reagents tobe used in a given reaction can be readily determined by the skilledartisan having the benefit of the current disclosure. The components ofthe kit may be present in separate containers, or multiple componentsmay be present in a single container.

The instructions for describing or practicing the methods of the presentinvention may be recorded on a suitable recording medium. For example,the instructions may be printed on a substrate, such as paper andplastic. In other embodiments, the instructions may be present as anelectronic storage data file present on a suitable computer readablestorage medium such as CD-ROM and diskette. In yet other embodiments,the actual instructions may not be present in the kit, but means forobtaining the instructions from a remote source, e.g. via the internet,are provided. An example of this embodiment is a kit that includes a webaddress where the instructions can be viewed and/or from which theinstructions can be downloaded.

IV. Storage Medium and Device for Differentiating Signals of Interestfor Target Nucleic Acid Sequences

Since the storage medium, the device and the computer program of theprevent invention described hereinbelow are intended to perform thepresent methods in a computer, the common descriptions between them areomitted in order to avoid undue redundancy leading to the complexity ofthis specification.

In still further aspect of this invention, there is provided a computerreadable storage medium containing instructions to configure a processorto perform a method for differentiating signals of interest for each oftwo target nucleic acid sequences comprising a first target nucleic acidsequence (T1) and a second target nucleic acid sequence (T2) in asample, which are not differentiable by a single type of detector, themethod comprising:

(a) receiving signals detected at a first detection temperature (D1) anda second detection temperature (D2); wherein the first target nucleicacid sequence (T1) in the sample is detected by a firstsignal-generating means and the second target nucleic acid sequence (T2)in the sample is detected by a second signal-generating means; whereinthe signals of interest to be generated by the two signal-generatingmeans are not differentiated by a single type of detector;

(b) providing the following two equations each of which comprisesvariables representing the signals of interest generated at eachdetection temperature for the two target nucleic acid sequences;S _(T1D1) +S _(T1D1) =S _(D1)  (I)S _(T1D2) +S _(T2D2) =S _(D2)  (II)

wherein (S_(D1)) is a signal detected at the first detectiontemperature, (S_(D2)) is a signal detected at the second detectiontemperature; (S_(T1D1)) is a variable representing a signal of interestgenerated by the first signal-generating means at the first detectiontemperature, (S_(T2D1)) is a variable representing a signal of interestgenerated by the second signal-generating means at the first detectiontemperature, (S_(T1D2)) is a variable representing a signal of interestgenerated by the first signal-generating means at the second detectiontemperature, (S_(T2D2)) is a variable representing a signal of interestgenerated by the second signal-generating means at the second detectiontemperature; and the total number of variables is four;

(c) providing two additional equations each of which comprises at leastone variable selected from the group consisting of the four variables,(S_(T1D1)), (S_(T2D1)), (S_(T1D2)) and (S_(T1D2)); and

(d) obtaining solutions to at least one of the variables by the fourequations provided in the steps (b) and (c) for differentiating at leastone of the signals of interest to be assigned to at least one of the twotarget nucleic acid sequences.

According to an embodiment of the present invention, the equations (I)and (II) as well as the additional equations is stored in the computerreadable storage medium.

According to an embodiment of the present invention, RV_(T1(D1D2)) as areference value (RV) of the first target nucleic acid sequence (T1)and/or RV_(T2(D1D2)) as a reference value (RV) of the second targetnucleic acid sequence (T2) is stored in the computer readable storagemedium.

According to an embodiment of the present invention, the computerreadable storage medium contains instructions to provide the equations(I) and (II) as well as the additional equations. According to anembodiment of the present invention, the computer readable storagemedium contains instructions to input RV_(T1(D1D2)) and/or RV_(T2(D1D2))in performing the method. According to an embodiment of the presentinvention, the computer readable storage medium further containsinstructions to configure a processor to perform a method for obtainingRV_(T1(D1D2)) and/or RV_(T2(D1D2)).

In another aspect of this invention, there is provided a computerprogram to be stored on a computer readable storage medium to configurea processor to perform a method for differentiating signals of interestfor each of two target nucleic acid sequences comprising a first targetnucleic acid sequence (T1) and a second target nucleic acid sequence(T2) in a sample, which are not differentiable by a single type ofdetector, the method comprising:

(a) receiving signals detected at a first detection temperature (D1) anda second detection temperature (D2); wherein the first target nucleicacid sequence (T1) in the sample is detected by a firstsignal-generating means and the second target nucleic acid sequence (T2)in the sample is detected by a second signal-generating means; whereinthe signals of interest to be generated by the two signal-generatingmeans are not differentiated by a single type of detector;

(b) providing the following two equations each of which comprisesvariables representing the signals of interest generated at eachdetection temperature for the two target nucleic acid sequences;S _(T1D1) +S _(T2D1) =S _(D1)  (I)S _(T1D2) +S _(T2D2) =S _(D2)  (II)

wherein (S_(D1)) is a signal detected at the first detectiontemperature, (S_(D2)) is a signal detected at the second detectiontemperature; (S_(T1D1)) is a variable representing a signal of interestgenerated by the first signal-generating means at the first detectiontemperature, (S_(T2D1)) is a variable representing a signal of interestgenerated by the second signal-generating means at the first detectiontemperature, (S_(T1D2)) is a variable representing a signal of interestgenerated by the first signal-generating means at the second detectiontemperature, (S_(T2D2)) is a variable representing a signal of interestgenerated by the second signal-generating means at the second detectiontemperature; and the total number of variables is four;

(c) providing two additional equations each of which comprises at leastone variable selected from the group consisting of the four variables,(S_(T1D1)), (S_(T2D1)), (S_(T1D2)) and (S_(T1D2)); and

(d) obtaining solutions to at least one of the variables by the fourequations provided in the steps (b) and (c) for differentiating at leastone of the signals of interest to be assigned to at least one of the twotarget nucleic acid sequences.

According to an embodiment of the present invention, the computerprogram contains the equations (I) and (II) as well as the additionalequations. According to an embodiment of the present invention, thecomputer program contains RV_(T1(D1D2)) and/or RV_(T2(D1D2)). Accordingto an embodiment of the present invention, the computer program containsinstructions to provide the equations (I) and (II) as well as theadditional equations. According to an embodiment of the presentinvention, the computer program contains instructions to inputRV_(T1(D1D2)) and/or RV_(T2(D1D2)) in performing the method. Accordingto an embodiment of the present invention, the computer program furthercontains instructions to configure a processor to perform a method forobtaining RV_(T1(D1D2)) and/or RV_(T2(D1D2)).

The program instructions are operative, when preformed by the processor,to cause the processor to perform the present method described above.The program instructions may comprise an instruction to receive signalsdetected at a first detection temperature (D1) and a second detectiontemperature (D2), and an instruction to obtain solutions to at least oneof the variables by using the four equations provided in the steps (b)and (c) for differentiating at least one of the signals of interest tobe assigned to at least one of the two target nucleic acid sequences.

The present method described above is implemented in a processor, suchas a processor in a stand-alone computer, a network attached computer ora data acquisition device such as a real-time PCR machine.

The types of the computer readable storage medium include variousstorage medium such as CD-R, CD-ROM, DVD, flash memory, floppy disk,hard drive, portable HDD, USB, magnetic tape, MINIDISC, nonvolatilememory card, EEPROM, optical disk, optical storage medium, RAM, ROM,system memory and web server.

The data (e.g., intensity, amplification cycle number and detectiontemperature) associated with the signals may be received through severalmechanisms. For example, the data may be acquired by a processorresident in a PCR data acquiring device. The data may be provided to theprocessor in real time as the data is being collected, or it may bestored in a memory unit or buffer and provided to the processor afterthe experiment has been completed. Similarly, the data set may beprovided to a separate system such as a desktop computer system via anetwork connection (e.g., LAN, VPN, intranet and Internet) or directconnection (e.g., USB or other direct wired or wireless connection) tothe acquiring device, or provided on a portable medium such as a CD,DVD, floppy disk, portable HDD or the like to a stand-alone computersystem. Similarly, the data set may be provided to a server system via anetwork connection (e.g., LAN, VPN, intranet, Internet and wirelesscommunication network) to a client such as a notebook or a desktopcomputer system. After the data has been received or acquired, the dataanalysis process proceeds to obtain solutions to at least one of thevariables by using the four equations provided in the steps (b) and (c)for differentiating at least one of the signals of interest to beassigned to at least one of the two target nucleic acid sequences. Forexample, the processor processes the received data to obtain adifference between the signals detected at the first detectiontemperature and the second detection temperature. The instructions toconfigure the processor to perform the present invention may be includedin a logic system. The instructions may be downloaded and stored in amemory module (e.g., hard drive or other memory such as a local orattached RAM or ROM), although the instructions can be provided on anysoftware storage medium such as a portable HDD, USB, floppy disk, CD andDVD. A computer code for implementing the present invention may beimplemented in a variety of coding languages such as C, C++, Java,Visual Basic, VBScript, JavaScript, Perl and XML. In addition, a varietyof languages and protocols may be used in external and internal storageand transmission of data and commands according to the presentinvention.

In still another aspect of this invention, there is provided a devicefor differentiating signals of interest for each of two target nucleicacid sequences comprising a first target nucleic acid sequence (T1) anda second target nucleic acid sequence (T2) in a sample, which are notdifferentiable by a single type of detector, comprising (a) a computerprocessor and (b) the computer readable storage medium described abovecoupled to the computer processor.

According to an embodiment, the device further comprises a reactionvessel to accommodate the sample and signal-generating means, atemperature controlling means to control temperatures of the reactionvessel and/or a single type detector to detect signals at the firstdetection temperature and the second detection temperature.

According to an embodiment, the computer processor permits not only thesingle type of detector to detect signals at the first detectiontemperature and the second detection temperature but also to obtainsolutions to at least one of the variables. The processor may beprepared in such a manner that a single processor can do twoperformances: direction of detection at two detection temperatures andcalculation of the solutions. Alternatively, the processor unit may beprepared in such a manner that two processors do two performances,respectively.

The first essential feature of the device carries the processor topermit the device to detect signals at the two detection temperatures.According to an embodiment, where the signal is generated along withamplification of the target nucleic acid sequence, the device comprisesa processor to permit the device to detect signals at the two detectiontemperatures at each amplification cycle.

The second essential feature of the device is to carry the processor toprocess signals at the two detection temperatures to obtain solutions tothe variables. According to an embodiment, the solutions to thevariables are expressed as numeric values by a mathematical processing.

According to an embodiment, the processor may be embodied by installingsoftware into conventional devices for detection of target nucleic acidsequences (e.g. real-time PCR device). According to an embodiment, thedevice comprises a processor to permit the device to detect signals attwo detection temperatures and to mathematically process two detectionresults.

In further aspect of this invention, there is provided a computerreadable storage medium containing instructions to configure a processorto perform a method for differentiating signals of interest for each oftarget nucleic acid sequences in the number of N in a sample, which arenot differentiable by a single type of detector, the method comprising:

(a) receiving signals detected at detection temperatures in the numberof N; wherein each of the target nucleic acid sequences in the sample isdetected by a corresponding signal-generating means; wherein the signalsof interest to be generated by the signal-generating means in the numberof N are not differentiated for each target nucleic acid sequence by asingle type of detector; wherein N is an integer not less than 2;

(b) providing the following equations in the number of N each of whichcomprises variables representing the signals of interest generated ateach detection temperature for the target nucleic acid sequences;

$\begin{matrix}{{S_{T\; 1D\; 1} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 1}}}} = S_{D\; 1}} & (1) \\{{S_{T\; 1D\; 2} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 2}}}} = {S_{D\; 2}\mspace{76mu} \cdot \mspace{275mu} \cdot \mspace{76mu} \cdot \mspace{275mu} \cdot \mspace{76mu} \cdot \mspace{275mu} \cdot}} & (2) \\{{S_{T\; 1{DN}} + S_{T\; 2{DN}} + {\cdot \cdot \cdot \cdot \cdot {+ S_{TNDN}}}} = S_{DN}} & (N)\end{matrix}$

wherein each of (S_(D1)) to (S_(DN)) is a signal detected at eachdetection temperature; each of (S_(T1D1)) to (S_(TNDN)) is a variablerepresenting a signal of interest generated by each signal-generatingmeans at each detection temperature; and the total number of thevariables is N²;

(c) providing additional equations in the number of (N²−N) each of whichcomprises at least one variable selected from the group consisting ofthe variables, (S_(T1D1)) to (S_(TNDN)); and

(d) obtaining solutions to at least one of the variables by theequations in the number of N² provided in the steps (b) and (c) fordifferentiating at least one of the signals of interest to be assignedto at least one of the target nucleic acid sequences in the number of N.

According to an embodiment of the present invention, the equations (1)to (N) as well as the additional equations is stored in the computerreadable storage medium. According to an embodiment of the presentinvention, RV_(Tj(DαDβ)) as a reference value is stored in the computerreadable storage medium.

According to an embodiment of the present invention, the computerreadable storage medium contains instructions to provide the equations(1) to (N) as well as the additional equations. According to anembodiment of the present invention, the computer readable storagemedium contains instructions to input RV_(Tj(DαDβ)) in performing themethod. According to an embodiment of the present invention, thecomputer readable storage medium further contains instructions toconfigure a processor to perform a method for obtaining RV_(Tj(DαDβ)).

In still further aspect of this invention, there is provided a computerprogram to be stored on a computer readable storage medium to configurea processor to perform a method for differentiating signals of interestfor each of target nucleic acid sequences in the number of N in asample, which are not differentiable by a single type of detector, themethod comprising:

(a) receiving signals detected at detection temperatures in the numberof N; wherein each of the target nucleic acid sequences in the sample isdetected by a corresponding signal-generating means; wherein the signalsof interest to be generated by the signal-generating means in the numberof N are not differentiated for each target nucleic acid sequence by asingle type of detector; wherein N is an integer not less than 2;

(b) providing the following equations in the number of N each of whichcomprises variables representing the signals of interest detected ateach detection temperature for the target nucleic acid sequences;

$\begin{matrix}{{S_{T\; 1D\; 1} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 1}}}} = S_{D\; 1}} & (1) \\{{S_{T\; 1D\; 2} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 2}}}} = {S_{D\; 2}\mspace{76mu} \cdot \mspace{275mu} \cdot \mspace{76mu} \cdot \mspace{275mu} \cdot \mspace{76mu} \cdot \mspace{275mu} \cdot}} & (2) \\{{S_{T\; 1{DN}} + S_{T\; 2{DN}} + {\cdot \cdot \cdot \cdot \cdot {+ S_{TNDN}}}} = S_{DN}} & (N)\end{matrix}$

wherein each of (S_(D1)) to (S_(DN)) is a signal detected at eachdetection temperature; each of (S_(T1D1)) to (S_(TNDN)) is a variablerepresenting a signal of interest provided by each signal-generatingmeans at each detection temperature; and the total number of thevariables is N²;

(c) providing additional equations in th7e number of (N²−N) each ofwhich comprises at least one variable selected from the group consistingof the variables, (S_(T1D1)) to (S_(TNDN)); and

(d) obtaining solutions to at least one of the variables by theequations in the number of N² provided in the steps (b) and (c) fordifferentiating at least one of the signals of interest to be assignedto at least one of the target nucleic acid sequences in the number of N.

According to an embodiment of the present invention, the computerprogram contains the equations (1) to (N) as well as the additionalequations. According to an embodiment of the present invention, thecomputer program contains RV_(Tj(DαDβ)). According to an embodiment ofthe present invention, the computer program contains instructions toprovide the equations (1) to (N) as well as the additional equations.According to an embodiment of the present invention, the computerprogram contains instructions to input RV_(Tj(DαDβ)) in performing themethod. According to an embodiment of the present invention, thecomputer program further contains instructions to configure a processorto perform a method for obtaining RV_(Tj(DαDβ)).

The program instructions are operative, when preformed by the processor,to cause the processor to perform the present method described above.The program instructions may comprise an instruction to receive signalsdetected at detection temperatures in the number of N, and aninstruction to obtain solutions to at least one of the variables fordifferentiating at least one of the signals of interest to be assignedto at least one of the target nucleic acid sequences in the number of N.

In another aspect of this invention, there is provided a device fordifferentiating signals of interest for each of target nucleic acidsequences in the number of N in a sample, which are not differentiableby a single type of detector, comprising (a) a computer processor and(b) the above-described computer readable storage medium coupled to thecomputer processor.

According to an embodiment, the device further comprises a reactionvessel to accommodate the sample and signal-generating means, atemperature controlling means to control temperatures of the reactionvessel and/or a single type detector to detect signals at detectiontemperatures in the number of N.

According to an embodiment, the computer processor permits not only thesingle type of detector to detect signals at detection temperatures inthe number of N but also to obtain solutions to at least one of thevariables. The processor may be prepared in such a manner that a singleprocessor can do two performances: direction of detection at detectiontemperatures in the number of N and calculation of the solutions.Alternatively, the processor unit may be prepared in such a manner thattwo processors do two performances, respectively.

The first essential feature of the device carries the processor topermit the device to detect signals at detection temperatures in thenumber of N. According to an embodiment, where the signal is generatedalong with amplification of the target nucleic acid sequence, the devicecomprises a processor to permit the device to detect signals atdetection temperatures in the number of N at each amplification cycle.

The second essential feature of the device is to carry the processor toprocess signals at detection temperatures in the number of N to obtainsolutions to the variables. According to an embodiment, the solutions tothe variables are expressed as numeric values by a mathematicalprocessing.

According to an embodiment, the processor may be embodied by installingsoftware into conventional devices for detection of target nucleic acidsequences (e.g. real-time PCR device). According to an embodiment, thedevice comprises a processor to permit the device to detect signals atdetection temperatures in the number of N and to mathematically processdetection results.

The features and advantages of this invention will be summarized asfollows:

(a) The present invention employing different detection temperaturesenables to detect a plurality of target nucleic acid sequences inconventional real-time manners even with a single type of label in asingle reaction vessel. The conventional technologies detect a pluralityof target nucleic acid sequences by a melting analysis after targetamplification. Unlikely, the present invention does not require amelting analysis after target amplification, such that the time foranalysis is greatly reduced.

(b) The present invention permits to obtain an individual signal value(i.e., variable) contained in a total signal detected at detectiontemperatures by using mathematical equations. The present inventionbased on equation-solving approach enables to obtain the individualsignal value in a systematical manner, thereby providing analysisresults in much more accurate and convenient manner.

(c) The present invention has a tremendous freedom in selectingsignal-generating means for detection of target nucleic acid sequences.In particular, the present invention may use (i) signal-generating meansto generate signals at all detection temperatures for each targetnucleic acid sequence, (ii) signal-generating means to generate signalsat all detection temperatures for a portion of target nucleic acidsequences and signal-generating means to generate signals at a portionof detection temperatures for a portion of target nucleic acidsequences, and (iii) signal-generating means with differentsignal-generating temperature range for different target nucleic acidsequences.

(d) The present invention embodies our findings in which there is acertain relationship (pattern or rule) in change of signals generated bysignal-generating means between two detection temperatures. Inparticular, the present invention introduces a novel concept “areference value representing such relationship (pattern or rule) inchange of signals” for detecting target nucleic acid sequences. Thepresent invention uses equations containing reference values. Theutilization of the reference value enables to provide the individualsignal value in much more logical manner and provide additionalequations in much more convenient manner.

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

EXAMPLES Example 1: Two Target Detection by Performing TaqMan Real-TimePCR Comprising Signal Detection at Different Temperatures and SolvingEquations

We examined whether two target nucleic acid sequences can be detected ina single reaction vessel by using a single detection channel and TaqManreal-time PCR comprising signal detection at different temperatures.Equations with variables representing signals of interest detected atthe two detection temperature were setup and solved for differentiatingthe signals of interest and determining the presence or absence oftarget nucleic acid sequences.

Taq DNA polymerase having a 5′ nuclease activity was used for theextension of upstream primers and downstream primers and the cleavage ofa TaqMan probe. Genomic DNA of Neisseria gonorrhoeae (NG) and genomicDNA of Chlamydia trachomatis (CT) were used as target nucleic acidsequences. Four types of samples (NG, CT, NG+CT and no template control)were prepared and analyzed.

TaqMan real-time PCR was employed to detect NG and CT. If a targetnucleic acid sequence is present, a TaqMan probe is cleaved and alabeled fragment is released. An amplification curve can be obtained bymeasuring a signal from the labeled fragment.

A TaqMan probe for NG is labeled with a fluorescent reporter molecule(Quasar 670) at its 5′-end and a quencher molecule at its 3′-end (SEQ IDNO: 3) and a TaqMan probe for CT with a fluorescent reporter molecule(Quasar 670) at its 5′-end and a quencher molecule in its inner part(BHQ-2) (SEQ ID NO: 6).

“60° C.” and “72° C.” were selected as signal detection temperatures.

The following equations were set up with variables representing signalsof interest provided by NG and CT at the 60° C. and 72° C. detectiontemperatures, reference values (RVs) of the NG and CT and signalsdetected at the two temperatures at every cycle during PCR.S _(T1D1) +S _(T2D1) =S _(D1)S _(T1D2) +S _(T2D2) =S _(D2)S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)  <Equation set 1>

S_(T1D1) is a variable representing a signal of interest provided by NGat 60° C. detection temperature wherein T1 represents NG, D1 represents60° C. detection temperature;

S_(T2D1) is a variable representing a signal of interest provided by CTat 60° C. detection temperature wherein T2 represents CT target, D1represents 60° C. detection temperature;

S_(T1D2) is a variable representing a signal of interest provided by NGat 72° C. detection temperature, wherein T1 represents NG, D2 represents72° C. detection temperature;

S_(T2D2) is a variable representing a signal of interest provided by CTat 72° C. detection temperature, wherein T2 represents CT, D2 represents72° C. detection temperature;

S_(D1) is a constant and obtained experimentally by measuring a signalat 60° C.;

S_(D2) is a constant and obtained experimentally by measuring signals at72° C.;

RV_(T1D1/D2) is a constant and obtained experimentally by calculating(RFU at 60° C.÷RFU at 72° C. at the end-point) wherein RFUs are thosemeasured for NG solely containing-sample. T1 represents NG, and D1 andD2 represent 60° C. and 72° C. detection temperatures, respectively; and

RV_(T2D1/D2) is a constant and obtained experimentally by calculatingRFU at 60° C.÷RFU at 72° C. at the end-point wherein RFUs are thosemeasured for CT solely containing-sample. T2 represents CT target, andD1 and D2 represent 60° C. and 72° C. detection temperatures,respectively.

One of approaches for solving the equation set is as follows: Linearsimultaneous equation with two variables each of which is selected foreach target sequence is newly established and then solutions to the twovariables are obtained. For instance, the following four linearsimultaneous equations may be set up:S _(T1D1) +S _(T2D1) =S _(D1)1/RV_(T1D1/D2) ×S _(T1D1)+1/RV_(T2D1/D2) ×S _(T2D1) =S_(D2)  Simultaneous equation 1RV_(T1D1/D2) ×S _(T1D2)+RV_(T2D1/D2) ×S _(T2D2) =S _(D1)S _(T1D2) +S _(T2D2) =S _(D2)  Simultaneous equation 2S _(T1D1)+RV_(T2D1/D2) ×S _(T2D2) =S _(D1)1/RV_(T1D1/D2) ×S _(T1D1) +S _(T2D2) =S _(D2)  Simultaneous equation 3RV_(T1D1/D2) ×S _(T1D2) +S _(T2D1) =S _(D1)S _(T1D2)+1/RV_(T2D1/D2) ×S _(T2D1) =S _(D2)  Simultaneous equation 4

Afterwards, one of the four linear simultaneous equations is selected.The solutions to a corresponding variable at each cycle are obtained andthen plotted. The plotting results may be considered as an amplificationcurve of a target sequence represented by a corresponding variable.Finally, the presence or absence of a target sequence is determined.

In this Example, the simultaneous equations 1 and 2 are employed.

The sequences of upstream primer, downstream primer, and probe used inthis Example are:

NG-F (SEQ ID NO: 1) 5′-TACGCCTGCTACTTTCACGCTIIIIIGTAATCAGATG-3′ NG-R(SEQ ID NO: 2) 5′-CAATGGATCGGTATCACTCGCIIIIICGAGCAAGAAC-3′ NG-P(SEQ ID NO: 3) 5′-[Quasar 670]-GCCCCTCATTGGCGTGTTTCG[BHQ-2]-3′ CT-F1(SEQ ID NO: 4) 5′-TCCGAATGGATAAAGCGTGACIIIIIATGAACTCAC-3′ CT-R1(SEQ ID NO: 5) 5′-AACAATGAATCCTGAGCAAAGGIIIIICGTTAGAGTC-3′ CT-P(SEQ ID NO: 6) 5′-[Quasar 670]CATTGTAAAGA[T(BHQ-2)]ATGGTCTGCTTCGACCG[C3 spacer]-3′

The real-time PCR was conducted in the final volume of 20 μl containinga target nucleic acid sequence (1 pg of NG genomic DNA, 10 pg of CTgenomic DNA or a mixture of 1 pg of NG genomic DNA and 10 pg of CTgenomic DNA), 5 pmole of upstream primer (SEQ ID NO: 1) and 10 pmole ofdownstream primer (SEQ ID NO: 2) for NG target amplification, 1.5 pmoleof TaqMan probe (SEQ ID NO: 3), 5 pmole of upstream primer (SEQ ID NO:4) and 10 pmole of downstream primer (SEQ ID NO: 5) for CT targetamplification, 3 pmole of TaqMan probe (SEQ ID NO: 6), and 5 μl of 4×Master Mix (final, 200 uM dNTPs, 2 mM MgCl₂, 2 U of Taq DNA polymerase).The tubes containing the reaction mixture were placed in the real-timethermocycler (CFX96, Bio-Rad) for 5 min at 50° C., denatured for 15 minat 95° C. and subjected to 50 cycles of 30 sec at 95° C., 60 sec at 60°C., 30 sec at 72° C. Detection of a signal was performed at 60° C. and72° C. of each cycle.

As shown in FIG. 1A, signals were detected both at 60° C. and 72° C. inthe presence of NG, CT, or NG+CT. No signal was detected in the absenceof the target nucleic acid sequences. Reference values of each targetwere calculated using the signals of NG only sample or CT only sampleand shown in FIG. 1B. As shown in FIG. 1B, reference values for NG andCT target were 1.8 and 5.8, respectively.

FIG. 1C shows plotting results obtained by solving the simultaneousequation 1. The plotting result for S_(T1D1) can be considered as anamplification curve of NG at 60° C. and the plotting result for S_(T2D1)be as an amplification curve of CT at 60° C. Proper thresholds wereselected with referring to the result of NG sample and CT sample toverify the significance of the obtained amplification curves. As shownin FIG. 1C, the amplification curves of the NG or the CT at 60° C. canshow the presence or absence of target nucleic acid sequences in theeach sample.

FIG. 1D shows that the amplification curves of the NG or the CT at 72°C. can be obtained by solving the simultaneous equation 2, whereby, thepresence or absence of target nucleic acid sequences in the each samplecan be determined.

These results demonstrate that the approach of setting up and solvingequations containing variables, reference values and signals measured ateach detection temperature allows for extracting or differentiating thesignal of interest for each target sequence from signals detected ateach detection temperature.

Therefore, two target nucleic acid sequences can be detected in a singlereaction vessel by using a single detection channel and TaqMan real-timePCR comprising signal detection at different temperatures, addressingthat the present equation solving-approach permits to determine thepresence of target sequences in much more convenient and reliablemanner.

Example 2: Two Target Detection by Performing PTOCE Real-Time PCRComprising Signal Detection at Different Temperatures and SolvingEquations

We examined whether two target nucleic acid sequences can be detected ina single reaction vessel by using a single detection channel and PTOCEreal-time PCR comprising signal detection at different temperatures.Equations with variables representing signals of interest detected atthe two detection temperature were setup and solved for differentiatingthe signals of interest and determining the presence or absence of thetarget nucleic acid sequences.

Taq DNA polymerase having a 5′ nuclease activity was used for theextension of upstream primers and downstream primers, the cleavage ofPTO, and the extension of PTO fragment. Genomic DNA of Neisseriagonorrhoeae (NG) and genomic DNA of Chlamydia trachomatis (CT) were usedas target nucleic acid sequences. Four types of samples (NG, CT, NG+CTand no template control) were prepared and analyzed.

PTOCE real-time PCR was used to detect CT and NG. If a target ispresent, a PTO is cleaved and a PTO fragment is produced. The PTOfragment is annealed to the capturing portion of the CTO, extended onthe templating portion of the CTO and forms an extended duplex with CTO(Duplexed CTO). The formation of the extended duplex provides a signaland an amplification curve can be obtained by measuring the signal atthe extended duplex-forming temperature.

“60° C.” and “72° C.” were selected as signal detection temperatures.

In this Example, the sequence and length of the extended duplex for CTis designed to provide a signal as it forms the duplex at 60° C. and 72°C. Meanwhile, the sequence and length of the extended duplex for NG isdesigned to provide a signal as it forms the duplex at 60° C., but notto provide a signal as it is dissociated not to forms the duplex at 72°C. In the detection temperature of 72° C., the signal for CT will begenerated and detected. In the detection temperature of 60° C., thesignal for NG as well as the signal for CT will be generated anddetected.

The PTO and CTO are blocked with a carbon spacer at their 3′-ends toprohibit their extension. The CTO is labeled with a quencher molecule(BHQ-2) and a fluorescent reporter molecule (CAL Fluor Red 610) in itstemplating portion (SEQ ID NOs: 8 and 12).S _(T1D1) +S _(T2D1) =S _(D1)S _(T1D2) +S _(T2D2) =S _(D2)S _(T1D2)=0S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)  <Equation set 2>

S_(T1D1), S_(T2D1), S_(T1D2), S_(T2D2), S_(D1), S_(D2) and RV_(T2D1/D2)are defined as those in the Equation set 1.

In the Equation set 2, the equation S_(T1D2)=0 is used instead ofRV_(T1D1/D2) as a reference value of NG. Because the signal-generatingmeans for NG was prepared to generate substantially no signal even inthe presence of NG at the 72° C. detection temperature (D2), theequation S_(T1D2)=0 can be adopted in this Example.

The solutions to a corresponding variable at each cycle are obtained andthen plotted.S _(T1D1) +S _(T2D1) =S _(D1)S _(T1D2) +S _(T2D2) =S _(D2)S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)  <Equation set 3>

S_(T1D1), S_(T2D1), S_(T1D2), S_(T2D2), S_(D1), S_(D2), RV_(T1D1/D2) andRV_(T2D1/D2) are defined as those in the Equation set 1.

In this Example, the signal-generating means for NG was prepared togenerate substantially no signal even in the presence of NG at the 72°C. detection temperature (D2). Nevertheless, a very weak signal (e.g.,background signal) detected at the 72° C. detection temperature (D2)which is usually detected in practical experiments may be used topractically provide RV_(T1D1/D2).

The solutions by using the Simultaneous equations 1 and 2 were obtainedin the same manner as solving of the Equation set 1. The solutions to acorresponding variable at each cycle were obtained and then plotted.S _(T1D1) +S _(T2D1) =S _(D1)S _(T1D2) +S _(T2D2) =S _(D2)S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)  <Equation set 4>

S_(T1D1), S_(T2D1), S_(T1D2), S_(T2D2), S_(D1), S_(D2), and RV_(T2D1/D2)are defined as those in the Equation set 1.

In the Equation set 4, RV_(T1D1/D2) is an arbitrary-selected constantvalue. In this Example, because the signal-generating means for NG wasprepared to generate substantially no signal even in the presence of NGat the 72° C. detection temperature (D2), an arbitrary-selected valuefor RV_(T1D1/D2) instead of experimentally-obtained values may be used.In this case, the arbitrary-selected value for RV_(T1D1/D2) is 10⁸ whichis much greater than experimentally-obtained values in the Equation set3.

The solutions by using the Simultaneous equations 1 and 2 were obtainedin the same manner as solving of the Equation set 1. The solutions to acorresponding variable at each cycle were obtained and then plotted.

The sequences of upstream primer, downstream primer, PTO, and CTO usedin this Example are:

NG-F (SEQ ID NO: 1) 5′-TACGCCTGCTACTTTCACGCTIIIIIGTAATCAGATG-3′ NG-R(SEQ ID NO: 2) 5′-CAATGGATCGGTATCACTCGCIIIIICGAGCAAGAAC-3′ NG-PTO(SEQ ID NO: 7) 5′-GTACGCGATACGGGCCCCTCATTGGCGTGTTTCG[C3 spacer]- 3′NG-CTO (SEQ ID NO: 8) 5′-[BHQ-2]TTTTTTTTTTTTTTTTTTTG[T(CAL Fluor Red610)]ACTGCCCGTATCGCGTAC[C3 spacer]-3′ CT-F2 (SEQ ID NO: 9)5′-GAGTTTTAAAATGGGAAATTCTGGTIIIIITTTGTATAAC-3′ CT-R2 (SEQ ID NO: 10)5′-CCAATTGTAATAGAAGCATTGGTTGIIIIITTATTGGAGA-3′ CT-PTO (SEQ ID NO: 11)5′-GATTACGCGACCGCATCAGAAGCTGTCATTTTGGCTGCG [C3 spacer]-3′ CT-CTO(SEQ ID NO: 12) 5′-[BHQ-2]GCGCTGGATACCCTGGACGA[T(Cal Fluor Red610)]ATGTGCGGTCGCGTAATC[C3 spacer]-3′ (I: Deoxyinosine) (Underlinedletters indicate the 5′-tagging portion of PTO)

The real-time PCR was conducted in the final volume of 20 μl containinga target nucleic acid sequence (10 pg of NG genomic DNA, 10 pg of CTgenomic DNA or a mixture of 10 pg of NG genomic DNA and 10 pg of CTgenomic DNA), 5 pmole of upstream primer (SEQ ID NO: 1) and 5 pmole ofdownstream primer (SEQ ID NO: 2) for NG target amplification, 3 pmole ofPTO (SEQ ID NO: 7), 1 pmole of CTO (SEQ ID NO: 8), 5 pmole of upstreamprimer (SEQ ID NO: 9) and 5 pmole of downstream primer (SEQ ID NO: 10)for CT target amplification, 3 pmole of PTO (SEQ ID NO: 11), 1 pmole ofCTO (SEQ ID NO: 12), and 10 μl of 2× Master Mix [final, 200 uM dNTPs, 2mM MgCl₂, 2 U of Taq DNA polymerase]. The tubes containing the reactionmixture were placed in the real-time thermocycler (CFX96, Bio-Rad) for 5min at 50° C., denatured for 15 min at 95° C. and subjected to 50 cyclesof 30 sec at 95° C., 60 sec at 60° C., 30 sec at 72° C. Detection of asignal was performed at 60° C. and 72° C. of each cycle.

As shown in FIG. 2A, signals were detected at 60° C. in the presence ofNG, CT, or NG+CT. In the sole presence of CT, a signal was detected bothat 60° C. and 72° C. In the sole presence of NG, a signal was detectedat 60° C. but not at 72° C. No signal was detected in the absence of thetarget nucleic acid sequences. Reference values of each target werecalculated using the signals of NG sample or CT sample and shown in FIG.2B. As shown in FIG. 2B, reference values for NG and CT target obtainedexperimentally were 36.7 and 1.2, respectively.

FIGS. 2C-2H show plotting results obtained by solving the equation set2, 3 and 4 respectively. The plotting result for S_(T1D1), S_(T2D1),S_(T1D2), S_(T2D2) can be considered as an amplification curve of NG at60° C., an amplification curve of CT at 60° C., an amplification curveof NG at 72° C. and an amplification curve of CT at 72° C.,respectively. Proper thresholds were selected with referring to theresult of NG sample and CT sample to verify the significance of theobtained amplification curves.

As shown in FIGS. 2C, 2E and 2G, the amplification curves of the NG orthe CT at 60° C. can confirm the presence or absence of target nucleicacid sequences in the each sample.

There results demonstrate that approach setting up and solving equationswith variables, reference values and signals measured at each detectiontemperature allows for extracting or differentiating the signal ofinterest for each target sequence from signals detected at eachdetection temperature.

Therefore, two target nucleic acid sequences can be detected in a singlereaction vessel by using a single detection channel and PTOCE real-timePCR comprising signal detection at different temperatures, addressingthat the present equation solving-approach permits to determine thepresence of target sequences in much more convenient and reliablemanner.

Example 3: Three Target Detection by Performing TaqMan/PTOCE Real-TimePCR Comprising Signal Detection at Different Temperatures and SolvingEquations

We examined whether triple target nucleic acid sequences can be detectedin a single reaction vessel by using a single detection channel andTaqMan/PTOCE real-time PCR comprising signal detection at differenttemperatures. Equations with variables representing signals of interestdetected at the three detection temperature were setup and solved fordifferentiating the signals of interest and determining the presence orabsence of the target nucleic acid sequences.

Taq DNA polymerase having a 5′ nuclease activity was used for theextension of upstream primers and downstream primers, the cleavage ofTaqMan probe, the cleavage of PTO, and the extension of PTO fragment.Genomic DNA of Neisseria gonorrhoeae (NG), genomic DNA of Chlamydiatrachomatis (CT), and genomic DNA of Mycoplasma genitalium (MG) wereused as target nucleic acid sequences. Eight reaction tubes wereprepared containing NG, CT, MG, a mixture of NG and CT, a mixture of NGand MG, a mixture of CT and MG, a mixture of NG, CT and MG, and notarget control respectively.

TaqMan real-time PCR was employed to detect MG. PTOCE real-time PCR wasused to detect CT and NG.

“60° C.”, “72° C.” and “95° C.” were selected as signal detectiontemperatures.

In this Example, the sequence and length of the extended duplex for CTis designed to provide a signal at 60° C. and 72° C. but not at 95° C.Meanwhile, the sequence and length of the extended duplex for NG isdesigned to provide a signal at 60° C., but not to provide a signal at72° C. and 95° C. In the detection temperature of 95° C., the signal forMG will be generated and detected. In the detection temperature of 72°C., the signal for CT will be generated and detected as well as thesignal for MG. Also, in the detection temperature of 60° C., the signalfor NG will be generated and detected as well as the signal for MG andCT.

TaqMan probe is labeled with a fluorescent reporter molecule (CAL FluorRed 610) at its 5′-end and a quencher molecule at its 3′-end (BHQ-2)(SEQ ID NO: 15). The PTO and CTO are blocked with a carbon spacer attheir 3′-ends to prohibit their extension. CTO is labeled with aquencher molecule (BHQ-2) and a fluorescent reporter molecule (CAL FluorRed 610) in its templating portion (SEQ ID NOs: 8 and 12).

In this Example, plotting methods using reference values of each targetand the signals from three detection temperatures (95° C., 72° C., and60° C.) provides amplification curves indicating the presence of eachtarget nucleic acid sequence. Simultaneous equations were used asplotting methods and were applied for the extraction of each target'ssignal from each detection temperatures. Calculation for reference valueand simultaneous equations to obtain amplification curves for eachtarget nucleic acid sequence were as follows:S _(T1D1) +S _(T2D1) +S _(T3D1) =S _(D1)S _(T1D2) +S _(T2D2) +S _(T3D2) =S _(D2)S _(T1D3) +S _(T2D3) +S _(T3D3) =S _(D3)S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)S _(T1D1) /S _(T1D3)=RV_(T1D1/D3)S _(T1D2) /S _(T1D3)=RV_(T1D2/D3)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)S _(T2D1) /S _(T2D3)=RV_(T2D1/D3)S _(T2D2) /S _(T2D3)=RV_(T2D2/D3)S _(T3D1) /S _(T3D2)=RV_(T3D1/D2)S _(T3D1) /S _(T3D3)=RV_(T3D1/D3)S _(T3D2) /S _(T3D3)=RV_(T3D2/D3)  <Equation set 5>

S_(T1D1) is a variable representing a signal of interest provided by NGat 60° C. detection temperature, wherein T1 represents NG target, D1represents 60° C. detection temperature;

S_(T2D1) is a variable representing a signal of interest provided by CTat 60° C. detection temperature, wherein T2 represents CT target, D1represents 60° C. detection temperature;

S_(T3D1) is a variable representing a signal of interest provided by MGat 60° C. detection temperature, wherein T3 represents MG target, D1represents 60° C. detection temperature;

S_(T1D2) is a variable representing a signal of interest provided by NGat 72° C. detection temperature, wherein T1 represents NG, D2 represents72° C. detection temperature;

S_(T2D2) is a variable representing a signal of interest provided by CTat 72° C. detection temperature, wherein T2 represents CT target, D2represents 72° C. detection temperature;

S_(T3D2) is a variable representing a signal of interest provided by MGat 72° C. detection temperature, wherein T3 represents MG target, D2represents 72° C. detection temperature;

S_(T1D3) is a variable representing a signal of interest provided by NGat 95° C. detection temperature, wherein T1 represents NG target, D3represents 95° C. detection temperature;

S_(T2D3) is a variable representing a signal of interest provided by CTat 95° C. detection temperature, wherein T2 represents CT target, D3represents 95° C. detection temperature;

S_(T3D3) is a variable representing a signal of interest provided by MGtarget at 95° C. detection temperature, wherein T3 represents MG target,D3 represents 95° C. detection temperature;

S_(D1) is a constant and obtained experimentally by measuring a signalat 60° C.;

S_(D2) is a constant and obtained experimentally by measuring signals at72° C.;

S_(D3) is a constant and obtained experimentally by measuring signals at95° C.;

RV_(T1D1/D2) is a constant and obtained experimentally by calculatingRFU at 60° C.÷RFU at 72° C. at the end-point wherein RFUs are thosemeasured in NG solely containing-sample. T1 represents NG, and D1 and D2represent 60° C. and 72° C. detection temperatures, respectively;

RV_(T1D1/D3) is a constant and obtained experimentally by calculatingRFU at 60° C.÷RFU at 95° C. at the end-point wherein RFUs are thosemeasured in NG solely containing-sample. T1 represents NG, and D1 and D3represent 60° C. and 95° C. detection temperatures, respectively;

RV_(T1D2/D3) is a constant and obtained experimentally by calculatingRFU at 72° C.÷RFU at 95° C. at the end-point wherein RFUs are thosemeasured in NG solely containing-sample. T1 represents NG, and D2 and D3represent 72° C. and 95° C. detection temperatures, respectively;

RV_(T2D1/D2) is a constant and obtained experimentally by calculatingRFU at 60° C.÷RFU at 72° C. at the end-point wherein RFUs are thosemeasured in CT solely containing-sample. T2 represents CT, and D1 and D2represent 60° C. and 72° C. detection temperatures, respectively;

RV_(T2D1/D3) is a constant and obtained experimentally by calculatingRFU at 60° C.÷RFU at 95° C. at the end-point wherein RFUs are thosemeasured in CT solely containing-sample. T2 represents CT, and D1 and D3represent 60° C. and 95° C. detection temperatures, respectively;

RV_(T2D2/D3) is a constant and obtained experimentally by calculatingRFU at 72° C.÷RFU at 95° C. at the end-point wherein RFUs are thosemeasured in CT solely containing-sample. T2 represents CT, and D2 and D3represent 72° C. and 95° C. detection temperatures, respectively;

RV_(T3D1/D2) is a constant and obtained experimentally by calculatingRFU at 60° C.÷RFU at 72° C. at the end-point wherein RFUs are thosemeasured in MG solely containing-sample. T3 represents MG, and D1 and D2represent 60° C. and 72° C. detection temperatures, respectively;

RV_(T3D1/D3) is a constant and obtained experimentally by calculatingRFU at 60° C.÷RFU at 95° C. at the end-point wherein RFUs are thosemeasured in MG solely containing-sample. T3 represents MG, and D1 and D3represent 60° C. and 95° C. detection temperatures, respectively; and

RV_(T3D2/D3) is a constant and obtained experimentally by calculatingRFU at 72° C.÷RFU at 95° C. at the end-point wherein RFUs are thosemeasured in MG solely containing-sample. T3 represents MG, and D2 and D3represent 72° C. and 95° C. detection temperatures, respectively.

One of approaches for solving the equation set is as follows: Linearsimultaneous equation with three variables each of which is selected foreach target sequence is newly established and then solutions to thethree variables are obtained. For instance, by using suitable sixequations among the nine RV equations in the Equation set 5, thefollowing three linear simultaneous equations may be set up:S _(T1D1) +S _(T2D1) +S _(T3D1) =S _(D1)1/RV_(T1D1/D2) ×S _(T1D1)+1/RV_(T2D1/D2) ×S _(T2D1)+1/RV_(T3D1/D2) ×S_(T3D1) =S _(D2)1/RV_(T1D1/D3) ×S _(T1D1)+1/RV_(T2D1/D3) ×S _(T2D1)+1/RV_(T3D1/D3) ×S_(T3D1) =S _(D3)  Simultaneous equation 5RV_(T1D1/D2) ×S _(T1D2)+RV_(T2D1/D2) ×S _(T2D2)+RV_(T3D1/D2) ×S _(T3D2)=S _(D1)S _(T1D2) +S _(T2D2) +S _(T3D2) =S _(D2)1/RV_(T1D2/D3) ×S _(T1D2)+1/RV_(T2D2/D3) ×S _(T2D2)+1/RV_(T3D2/D3) ×S_(T3D2) =S _(D3)  Simultaneous equation 6RV_(T1D1/D3) ×S _(T1D3)+RV_(T2D1/D3) ×S _(T2D3)+RV_(T3D1/D3) ×S _(T3D3)=S _(D1)RV_(T1D2/D3) ×S _(T1D3)+RV_(T2D2/D3) ×S _(T2D3)+RV_(T3D2/D3) ×S _(T3D3)=S _(D2)S _(T1D3) +S _(T2D3) +S _(T3D3) =S _(D3)  Simultaneous equation 7

Afterwards, the solutions to a corresponding variable at each cycle areobtained and then plotted. The plotting results may be considered as anamplification curve of a target sequence represented by a correspondingvariable. Finally, the presence or absence of a target sequence isdetermined.

In this Example, the signal-generating means for NG was prepared togenerate substantially no signal even in the presence of NG at the 72°C. and 95° C. detection temperatures, and the signal-generating meansfor CT was prepared to generate substantially no signal even in thepresence of CT at the 95° C. detection temperature. Therefore, otherequation sets may be also used instead of the Equation set 5.

As a first modification example of the Equation set 5, the equationsS_(T1D2)=0 and S_(T1D3)=0 are used instead of equations comprisingRV_(T1D1/D2), RV_(T1D1/D3) or RV_(T1D2/D3) among reference values forNG, and the equation S_(T2D3)=0 is used instead of equations comprisingRV_(T2D1/D3) or RV_(T2D2/D3) among reference values for CT.

According to a second modification example, arbitrary-selected valuesfor RV_(T1D1/D2), RV_(T1D1/D3), RV_(T1D2/D3), RV_(T2D1/D3) andRV_(T2D2/D3) may be used instead of experimentally-obtained values.

A combinatory embodiment of the first and second modifications may bealso used in the present method.

The sequences of upstream primer, downstream primer, PTO, CTO and TaqManprobe used in this Example are:

NG-F (SEQ ID NO: 1) 5′-TACGCCTGCTACTTTCACGCTIIIIIGTAATCAGATG-3′ NG-R(SEQ ID NO: 2) 5′-CAATGGATCGGTATCACTCGCIIIIICGAGCAAGAAC-3′ NG-PTO(SEQ ID NO: 7) 5′-GTACGCGATACGGGCCCCTCATTGGCGTGTTTCG[C3 spacer]- 3′NG-CTO (SEQ ID NO: 8) 5′-[BHQ-2]TTTTTTTTTTTTTTTTTTTG[T(CAL Fluor Red610)]ACTGCCCGTATCGCGTAC[C3 spacer]-3′ CT-F2 (SEQ ID NO: 9)5′-GAGTTTTAAAATGGGAAATTCTGGTIIIIITTTGTATAAC-3′ CT-R2 (SEQ ID NO: 10)5′-CCAATTGTAATAGAAGCATTGGTTGIIIIITTATTGGAGA-3′ CT-PTO (SEQ ID NO: 11)5′-GATTACGCGACCGCATCAGAAGCTGTCATTTTGGCTGCG [C3 spacer]-3′ CT-CTO(SEQ ID NO: 12) 5′-[BHQ-2]GCGCTGGATACCCTGGACGA[T(Cal Fluor Red610)]ATGTGCGGTCGCGTAATC[C3 spacer]-3′ MG-F (SEQ ID NO: 13)5′-AAAACCCACGGAAATGATGAGAIIIIIATTGGTTCTAC-3′ MG-R (SEQ ID NO: 14)5′-CTCGTTAATTTACCTATTCCATTTTGIIIIICTGATAAAAG-3′ MG-P (SEQ ID NO: 15)5′-[CAL Fluor Red 610]GAGTTCTTTCAAGAACAGCAAGAGGT GT[BHQ-2]-3′ (I:Deoxyinosine) (Underlined letters indicate the 5′-tagging portion ofPTO)

The real-time PCR was conducted in the final volume of 20 μl containinga target nucleic acid sequence (10 pg of NG genomic DNA, 10 pg of CTgenomic DNA, 10 pg of MG genomic DNA, a mixture of each 10 pg of NG andCT genomic DNA, a mixture of each 10 pg of NG and MG genomic DNA, amixture of each 10 pg of CT and MG genomic DNA; or a mixture of each 10pg of NG, CT and MG genomic DNA), 5 pmole of upstream primer (SEQ IDNO: 1) and 5 pmole of downstream primer (SEQ ID NO: 2) for NG targetamplification, 3 pmole of PTO (SEQ ID NO: 7), 1 pmole of CTO (SEQ ID NO:8), 5 pmole of upstream primer (SEQ ID NO: 9) and 5 pmole of downstreamprimer (SEQ ID NO: 10) for CT target amplification, 3 pmole of PTO (SEQID NO: 11), 1 pmole of CTO (SEQ ID NO: 12), 5 pmole of upstream primer(SEQ ID NO: 13) and 5 pmole of downstream primer (SEQ ID NO: 14) for MGtarget amplification, 1 pmole of TaqMan probe (SEQ ID NO: 15), and 10 μlof 2× Master Mix [final, 200 uM dNTPs, 2 mM MgCl₂, 2 U of Taq DNApolymerase]. The tubes containing the reaction mixture were placed inthe real-time thermocycler (CFX96, Bio-Rad) for 5 min at 50° C.,denatured for 15 min at 95° C. and subjected to 50 cycles of 30 sec at95° C., 60 sec at 60° C., 30 sec at 72° C. Detection of a signal wasperformed at 60° C., 72° C., and 95° C. of each cycle.

As shown in FIGS. 3A and 3B, signals were detected at 60° C. in thepresence of NG, CT, MG, and mixed targets. In the sole presence of CT, asignal was detected at 72° C. and 60° C. but not at 95° C. In the solepresence of NG, a signal was detected at 60° C. but not at 95° C. and72° C. No signal was detected in the absence of the target nucleic acidsequences. Reference values of each target were calculated using thesignals of NG only sample or CT only or MG only sample and shown in FIG.3C.

The plotting result for S_(T1D1), S_(T2D1), S_(T3D1), S_(T1D2), S_(T2D2)S_(T3D2), S_(T1D3), S_(T2D3), and S_(T3D3) can be considered as anamplification curve of NG at 60° C., an amplification curve of CT at 60°C., an amplification curve of MG at 60° C., an amplification curve of NGat 72° C., an amplification curve of CT at 72° C., an amplificationcurve of MG at 72° C., an amplification curve of NG at 95° C., anamplification curve of CT at 95° C., and an amplification curve of MG at95° C. respectively. Proper thresholds were selected referring to theresult of NG only sample, CT only sample, and MG only sample to verifythe significance of the obtained amplification curves.

As shown in FIGS. 3D and 3E, the amplification curves of the NG, the CT,or the MG at 60° C. can confirm the presence or absence of targetnucleic acid sequences in the each sample.

These results demonstrate that approach setting up and solving equationswith variables, reference values and signals measured at each detectiontemperature allows for extracting or differentiating the signal ofinterest for each target sequence from signals detected at eachdetection temperature.

Therefore, three target nucleic acid sequences can be detected in asingle reaction vessel by using a single detection channel andTaqMan/PTOCE real-time PCR comprising signal detection at differenttemperatures, addressing that the present equation solving-approachpermits to determine the presence of at least three target sequences inmuch more convenient and reliable manner.

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

What is claimed is:
 1. A method for differentiating signals of interestfor each of two target nucleic acid sequences comprising a first targetnucleic acid sequence (T1) and a second target nucleic acid sequence(T2) in a sample, which are not differentiable by a single type ofdetector, comprising: (a) incubating the sample with a firstsignal-generating means for detection of the first target nucleic acidsequence (T1) and a second signal-generating means for detection of thesecond target nucleic acid sequence (T2) and detecting signals at afirst detection temperature (D1) and a second detection temperature(D2); wherein the signals of interest to be generated by the twosignal-generating means are not differentiated for each target nucleicacid sequence by a single type of detector; (b) providing the followingtwo equations each of which comprises variables representing the signalsof interest generated at each detection temperature for the two targetnucleic acid sequences;S _(T1D1) +S _(T2D1) =S _(D1)  (I)S _(T1D2) +S _(T2D2) =S _(D2)  (II) wherein (S_(D1)) is a signaldetected at the first detection temperature, (S_(D2)) is a signaldetected at the second detection temperature; (S_(T1D1)) is a variablerepresenting a signal of interest generated by the firstsignal-generating means at the first detection temperature, (S_(T2D1))is a variable representing a signal of interest generated by the secondsignal-generating means at the first detection temperature, (S_(T1D2))is a variable representing a signal of interest generated by the firstsignal-generating means at the second detection temperature, (S_(T2D2))is a variable representing a signal of interest generated by the secondsignal-generating means at the second detection temperature; and thetotal number of variables is four; (c) providing two additionalequations selected from the group consisting of the following equationsf(S _(T1D1) ,S _(T1D2))=RV_(T1(D1D2))  (III),f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2))  (IV), andS _(T1D2)=an arbitrarily selected value  (V) wherein, RV_(T1(D1D2)) is areference value (RV) of the first target nucleic acid sequence (T1)representing a relationship of change in signals provided by the firstsignal-generating means at the first detection temperature and thesecond detection temperature, RV_(T2(D1D2)) is a reference value (RV) ofthe second target nucleic acid sequence (T2) representing a relationshipof change in signals provided by the second signal-generating means atthe first detection temperature and the second detection temperature;f(S_(T1D1), S_(T1D2)) represents a function of S_(T1D1) and S_(T1D2);f(S_(T2D1), S_(T2D2)) represents a function of S_(T2D1) and S_(T2D2);wherein S_(T1D2)=an arbitrarily selected value in the equation (V) maybe selected with a proviso that the first signal-generating means isprepared to generate no signal in the presence of the first targetnucleic acid sequence at the second detection temperature; and (d)obtaining solutions to at least one of the variables by the fourequations provided in the steps (b) and (c) for differentiating at leastone of the signals of interest to be assigned to at least one of the twotarget nucleic acid sequences.
 2. The method of claim 1, wherein themethod is performed to detect at least one of the two target nucleicacid sequences in the sample by assigning the at least one of thesignals of interest to at least one of the two target nucleic acidsequences.
 3. The method of claim 1, wherein the equation (III) is usedto convert the two variables (S_(T1D1)) and (S_(T1D2)) in the equations(I) and (II) into a variable selected from the two variables, and theequation (IV) is used to convert the two variables (S_(T2D1)) and(S_(T2D2)) in the equations (I) and (II) into a variable selected fromthe two variables.
 4. The method of claim 1, wherein (i) RV_(T1(D1D2))is obtained by (i-1) incubating the first target nucleic acid sequencewith the first signal-generating means for detection of the first targetnucleic acid sequence, (i-2) detecting signals at the first detectiontemperature and the second detection temperature, and (i-3) thenobtaining a difference between the signals detected at the firstdetection temperature and the second detection temperature, and (ii)RV_(T2(D1D2)) is obtained by (ii-1) incubating the second target nucleicacid sequence with the second signal-generating means for detection ofthe second target nucleic acid sequence, (ii-2) detecting signals at thefirst detection temperature and the second detection temperature, and(ii-3) then obtaining a difference between the signals detected at thefirst detection temperature and the second detection temperature;wherein RV_(T1(D1D2)) is different from RV_(T2(D1D2)).
 5. The method ofclaim 1, wherein f(S_(T1D1), S_(T1D2))=RV_(T1(D1D2)) comprisesS_(T1D1)/S_(T1D2)=RV_(T1D1/D2) (VI) or S_(T1D2)/S_(T1D1)=RV_(T1D2/D1)(VII); f(S_(T2D1), S_(T2D2))=RV_(T2(D1D2)) comprisesS_(T2D1)/S_(T2D2)=RV_(T2D1/D2) (VIII) or S_(T2D2)/S_(T2D1)=RV_(T2D2/D1)(IX).
 6. The method of claim 5, wherein RV_(T1D1/D2) is obtained by(i-1) incubating the first target nucleic acid sequence with the firstsignal-generating means for detection of the first target nucleic acidsequence, (i-2) detecting signals at the first detection temperature andthe second detection temperature, and (i-3) then calculating a ratio ofa signal detected at the first detection temperature to a signaldetected at the second detection temperature; RV_(T1D2/D1) is obtainedby performing the steps (i-1) and (i-2) and then calculating a ratio ofthe signal detected at the second detection temperature to the signaldetected at the first detection temperature; RV_(T2D1/D2) is obtained by(ii-1) incubating the second target nucleic acid sequence with thesecond signal-generating means for detection of the second targetnucleic acid sequence, (ii-2) detecting signals at the first detectiontemperature and the second detection temperature, and then (ii-3)calculating a ratio of the signal detected at the first detectiontemperature to the signal detected at the second detection temperature;and RV_(T2D2/D1) is obtained by performing the steps (ii-1) and (ii-2)and then calculating a ratio of the signal detected at the seconddetection temperature to the signal detected at the first detectiontemperature.
 7. The method of claim 1, wherein when the firstsignal-generating means is prepared to generate no signal in thepresence of the first target nucleic acid sequence at the seconddetection temperature, RV_(T1(D1D2)) is an arbitrarily selected value.8. The method of claim 1, wherein the two additional equations forobtaining (S_(T1D1)), (S_(T1D2)), (S_(T2D1)) and (S_(T2D2)) comprisesone of the following equations (VI) and (VII) and one of the followingequations (VIII) and (IX):S _(T1D1) /S _(T1D2)=RV_(T1D1/D2)  (VI)S _(T1D2) /S _(T1D1)=RV_(T1D2/D1)  (VII)S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)  (VIII)S _(T2D2) /S _(T2D1)=RV_(T2D2/D1)  (IX).
 9. The method of claim 1,wherein the two additional equations for obtaining (S_(T1D1)),(S_(T1D2)), (S_(T2D1)) and (S_(T2D2)) comprises one of the followingequations (VIII) and (IX) and the following equation (V):S _(T2D1) /S _(T2D2)=RV_(T2D1/D2)  (VIII)S _(T2D2) /S _(T2D1)=RV_(T2D2/D1)  (IX)S _(T1D2)=an arbitrarily selected value  (V).
 10. A method fordifferentiating signals of interest for each of target nucleic acidsequences in the number of N in a sample, which are not differentiableby a single type of detector, comprising: (a) incubating the sample withsignal-generating means in the number of N for detection of the targetnucleic acid sequences in the number of N and detecting signals atdetection temperatures in the number of N; wherein each of the targetnucleic acid sequences is detected by a corresponding signal-generatingmeans; wherein the signals of interest to be generated by thesignal-generating means in the number of N are not differentiated foreach target nucleic acid sequence by a single type of detector; whereinN is an integer not less than 2; (b) providing the following equationsin the number of N each of which comprises variables representing thesignals of interest generated at each detection temperature for thetarget nucleic acid sequences: $\begin{matrix}{{S_{T\; 1D\; 1} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 1}}}} = S_{D\; 1}} & (1) \\{{S_{T\; 1D\; 2} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 2}}}} = {S_{D\; 2}\mspace{34mu} \cdot \cdot \mspace{76mu} \cdot \mspace{169mu} \cdot \mspace{34mu} \cdot \cdot}} & (2) \\{{S_{T\; 1{DN}} + S_{T\; 2{DN}} + {\cdot \cdot \cdot \cdot \cdot {+ S_{TNDN}}}} = S_{DN}} & (N)\end{matrix}$ wherein each of (S_(D1)) to (S_(DN)) is a signal detectedat each detection temperature; each of (S_(T1D1)) to (S_(TNDN)) is avariable representing a signal of interest generated by eachsignal-generating means at each detection temperature; and the totalnumber of the variables is N²; (c) providing additional equations in thenumber of (N²−N) selected from the group consisting of the followingequationsf(S _(TjDα) ,S _(TjDβ))=RV_(Tj(DαDβ))  (X), andS _(TiDK)=an arbitrarily selected value  (XI) wherein, j in Tjrepresents all integers starting from 1 to a N^(th) integer; for eachj^(th) target nucleic acid, α and β jointly represent a combination oftwo integers selected from 1 to N; wherein the number of the combinationis represented by _(N)C₂; for each j^(th) target nucleic acid, K is atleast one of integers selected from 1 to N; RV_(Tj(DαDβ)) is a referencevalue (RV) of the j^(th) target nucleic acid sequence (Tj) representingrelationship reflecting change in signals provided by the j^(th)signal-generating means at the α^(th) detection temperature and theβ^(th) detection temperature; f(S_(TjDα), S_(TjDβ)) represents afunction of S_(TjDα) and S_(TjDβ); wherein S_(TjDK)=an arbitrarilyselected value in the equation (XI) may be selected with a proviso thatthe j^(th) signal-generating means is prepared to generate no signal inthe presence of the j^(th) target nucleic acid sequence at the K^(th)detection temperature; and (d) obtaining solutions to at least one ofthe variables by the equations in the number of N² provided in the steps(b) and (c) for differentiating at least one of the signals of interestto be assigned to at least one of the target nucleic acid sequences inthe number of N.
 11. The method of claim 10, wherein the method isperformed to detect at least one of the target nucleic acid sequences inthe sample by assigning the at least one of the signals of interest toat least one of the target nucleic acid sequences.
 12. The method ofclaim 10, wherein the equation (X) comprising the reference value (RV)of the j^(th) target nucleic acid sequence (Tj) is used to convert thevariables (S_(TjD1)) to (S_(TjDN)) in the equations (1) to (N) into avariable selected from the variables (S_(TjD1)) to (S_(TjDN)).
 13. Themethod of claim 10, wherein RV_(Tj(DαDβ)) is obtained by (i−1)incubating the j^(th) target nucleic acid sequence with the j^(th)signal-generating means for detection of the j^(th) target nucleic acidsequence, (i−2) detecting signals at the α^(th) detection temperatureand the β^(th) detection temperature, and (i−3) then obtaining adifference between the signals detected at the α^(th) detectiontemperature and the β^(th) detection temperature.
 14. The method ofclaim 10, wherein f(S_(TjDα), S_(TjDβ))=RV_(Tj(DαDβ)) comprisesS_(TjDα)/S_(TjDβ)=RV_(TjDα/Dβ) (XII) or S_(TjDβ)/S_(TjDα)=RV_(TjDβ/Dα)(XIII).
 15. The method of claim 14, wherein RV_(TjDα/Dβ) is obtained by(i-1) incubating the j^(th) target nucleic acid sequence with the j^(th)signal-generating means for detection of the j^(th) target nucleic acidsequence, (i-2) detecting signals at the α^(th) detection temperatureand the β^(th) detection temperature, and (i-3) then calculating a ratioof a signal detected at the α^(th) detection temperature to a signaldetected at the β^(th) detection temperature; RV_(TjDβ/Dα) is obtainedby performing the steps (i-1) and (i-2) and then calculating a ratio ofthe signal detected at the β^(th) detection temperature to the signaldetected at the α^(th) detection temperature.
 16. The method of claim10, wherein when the j^(th) signal-generating means is prepared togenerate no signal in the presence of the j^(th) target nucleic acidsequence at the K^(th) detection temperature, RV_(Tj(DαDβ)) in which αor β is the same as K is an arbitrarily selected value.
 17. The methodof claim 10, wherein the additional equations in the number of N²−N areselected from the groups of the following equations:S _(TjDα) /S _(TjDβ)=RV_(TjDα/Dβ)  (XII)S _(TjDβ) /S _(TjDα)=RV_(TjDβ/Dα)  (XIII) wherein the equation for eachj^(th) target nucleic acid at the α^(th) detection temperature and theβ^(th) detection temperature is selected from the equations (XII) and(XIII).
 18. The method of claim 10, wherein the additional equations innumbers of N²−N are selected from the groups of the following equations:S _(TjDα) /S _(TjDβ)=RV_(TjDα/Dβ)  (XII)S _(TjDβ) /S _(TjDα)=RV_(TjDβ/Dα)  (XIII)S _(TjDK)=an arbitrarily selected value  (XI) wherein the equation foreach j^(th) target nucleic acid at the α^(th) detection temperature andthe β^(th) detection temperature is selected from the equations (XII)and (XIII); wherein when the equation (XI) is selected as the additionalequations, the other additional equations are selected from theequations (XII) and (XIII) in which α or β for the j^(th) target nucleicacid sequence is different from K for the j^(th) target nucleic acidsequence.
 19. The method of claim 1, wherein the step (a) is performedin a signal amplification process concomitantly with a nucleic acidamplification.
 20. The method of claim 1, wherein the step (a) isperformed in a signal amplification process without a nucleic acidamplification.
 21. A kit for differentiating signals of interest foreach of two target nucleic acid sequences comprising a first targetnucleic acid sequence (T1) and a second target nucleic acid sequence(T2) in a sample, which are not differentiable by a single type ofdetector, comprising: (a) two signal-generating means for detection ofthe two target nucleic acid sequences; and (b) an instruction thatdescribes the method of claim
 1. 22. A kit for differentiating signalsof interest for each of target nucleic acid sequences in the number of Nin a sample, which are not differentiable by a single type of detector,comprising: (a) signal-generating means in the number of N for detectionof the target nucleic acid sequences in the number of N; and (b) aninstruction that describes the method of claim
 10. 23. A non-transitorycomputer readable storage medium containing instructions to configure aprocessor to perform a method for differentiating signals of interestfor each of two target nucleic acid sequences comprising a first targetnucleic acid sequence (T1) and a second target nucleic acid sequence(T2) in a sample, which are not differentiable by a single type ofdetector, the method comprising: (a) receiving signals detected at afirst detection temperature (D1) and a second detection temperature(D2); wherein the first target nucleic acid sequence (T1) in the sampleis detected by a first signal-generating means and the second targetnucleic acid sequence (T2) in the sample is detected by a secondsignal-generating means; wherein the signals of interest to be generatedby the two signal-generating means are not differentiated by a singletype of detector; (b) providing the following two equations each ofwhich comprises variables representing the signals of interest generatedat each detection temperature for the two target nucleic acid sequences;S _(T1D1) +S _(T2D1) =S _(D1)  (I)S _(T1D2) +S _(T2D2) =S _(D2)  (II) wherein (S_(D1)) is a signaldetected at the first detection temperature, (S_(D2)) is a signaldetected at the second detection temperature; (S_(T1D1)) is a variablerepresenting a signal of interest generated by the firstsignal-generating means at the first detection temperature, (S_(T2D1))is a variable representing a signal of interest generated by the secondsignal-generating means at the first detection temperature, (S_(T1D2))is a variable representing a signal of interest generated by the firstsignal-generating means at the second detection temperature, (S_(T2D2))is a variable representing a signal of interest generated by the secondsignal-generating means at the second detection temperature; and thetotal number of variables is four; (c) providing two additionalequations selected from the group consisting of the following equationsf(S _(T1D1) ,S _(T1D2))=RV_(T1(D1D2))  (III),f(S _(T2D1) ,S _(T2D2))=RV_(T2(D1D2))  (IV), andS _(T1D2)=an arbitrarily selected value  (V) wherein, RV_(T1(D1D2)) is areference value (RV) of the first target nucleic acid sequence (T1)representing a relationship of change in signals provided by the firstsignal-generating means at the first detection temperature and thesecond detection temperature, RV_(T2(D1D2)) is a reference value (RV) ofthe second target nucleic acid sequence (T2) representing a relationshipof change in signals provided by the second signal-generating means atthe first detection temperature and the second detection temperature;f(S_(T1D1), S_(T1D2)) represents a function of S_(T1D1) and S_(T1D2);f(S_(T2D1), S_(T2D2)) represents a function of S_(T2D1) and S_(T2D2);wherein S_(T1D2)=an arbitrarily selected value in the equation (V) maybe selected with a proviso that the first signal-generating means isprepared to generate no signal in the presence of the first targetnucleic acid sequence at the second detection temperature whereinf(S_(T1D1), S_(T1D2))=RV_(T1(D1D2)) is S_(T1D1)/S_(T1D2)=RV_(T1D1/D2)(VI) or S_(T1D2)/S_(T1D1)=RV_(T1D2/D1) (VII); f(S_(T2D1),S_(T2D2))=RV_(T2(D1D2)) is S_(T2D1)/S_(T2D2)=RV_(T2D1/D2) (VIII) orS_(T2D2)/S_(T2D1)=RV_(T2D2/D1) (IX); and (d) obtaining solutions to atleast one of the variables by the four equations provided in the steps(b) and (c) for differentiating at least one of the signals of interestto be assigned to at least one of the two target nucleic acid sequences.24. A non-transitory computer readable storage medium containinginstructions to configure a processor to perform a method fordifferentiating signals of interest for each of target nucleic acidsequences in the number of N in a sample, which are not differentiableby a single type of detector, the method comprising: (a) receivingsignals detected at detection temperatures in the number of N; whereineach of the target nucleic acid sequences in the sample is detected by acorresponding signal-generating means; wherein the signals of interestto be generated by the signal-generating means in the number of N arenot differentiated for each target nucleic acid sequence by a singletype of detector; wherein N is an integer not less than 2; (b) providingthe following equations in the number of N each of which comprisesvariables representing the signals of interest generated at eachdetection temperature for the target nucleic acid sequences;$\begin{matrix}{{S_{T\; 1D\; 1} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 1}}}} = S_{D\; 1}} & (1) \\{{S_{T\; 1D\; 2} + S_{T\; 2D\; 2} + {\cdot \cdot \cdot \cdot \cdot {+ S_{{TND}\; 2}}}} = {S_{D\; 2}\mspace{34mu} \cdot \cdot \mspace{76mu} \cdot \mspace{169mu} \cdot \mspace{34mu} \cdot \cdot}} & (2) \\{{S_{T\; 1{DN}} + S_{T\; 2{DN}} + {\cdot \cdot \cdot \cdot \cdot {+ S_{TNDN}}}} = S_{DN}} & (N)\end{matrix}$ wherein each of (S_(D1)) to (S_(DN)) is a signal detectedat each detection temperature; each of (S_(T1D1)) to (S_(TNDN)) is avariable representing a signal of interest generated by eachsignal-generating means at each detection temperature; and the totalnumber of the variables is N²; (c) providing additional equations in thenumber of (N²−N) selected from the group consisting of the followingequations:f(S _(TjDα) ,S _(TjDβ))=RV_(Tj(DαDβ))  (X), andS _(TjDK)=an arbitrarily selected value  (XI) wherein, j in Tjrepresents all integers starting from 1 to a N^(th) integer; for eachj^(th) target nucleic acid, α and β jointly represent a combination oftwo integers selected from 1 to N; wherein the number of the combinationis represented by _(N)C₂; for each j^(th) target nucleic acid, K is atleast one of integers selected from 1 to N; RV_(Tj(DαDβ)) is a referencevalue (RV) of the j^(th) target nucleic acid sequence (Tj) representingrelationship reflecting change in signals provided by the j^(th)signal-generating means at the α^(th) detection temperature and theβ^(th) detection temperature; f(S_(TjDα), S_(TjDβ)) represents afunction of S_(TjDα) and S_(TjDβ); wherein S_(TjDK)=an arbitrarilyselected value in the equation (XI) may be selected with a proviso thatthe j^(th) signal-generating means is prepared to generate no signal inthe presence of the j^(th) target nucleic acid sequence at the K^(th)detection temperature wherein f(S_(TjDα), S_(TjDβ))=RV_(Tj(DαDβ)) isS_(TjDα)/S_(TjDβ)=RV_(TjDα/Dβ) (XII) or S_(TjDβ)/S_(TjDα)=RV_(TjDβ/Dα)(XIII); and (d) obtaining solutions to at least one of the variables bythe equations in the number of N² provided in the steps (b) and (c) fordifferentiating at least one of the signals of interest to be assignedto at least one of the target nucleic acid sequences in the number of N.25. A device for differentiating signals of interest for each of twotarget nucleic acid sequences comprising a first target nucleic acidsequence (T1) and a second target nucleic acid sequence (T2) in asample, which are not differentiable by a single type of detector,comprising (a) a computer processor and (b) the computer readablestorage medium of claim 23 coupled to the computer processor.
 26. Adevice for differentiating signals of interest for each of targetnucleic acid sequences in the number of N in a sample, which are notdifferentiable by a single type of detector, comprising (a) a computerprocessor and (b) the computer readable storage medium of claim 24coupled to the computer processor.